bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The trafficking protein Ambra1 regulates transcription

A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes

Christina Schoenherr1, Adam Byron1 and Margaret C Frame1* 1Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, EH4 2XR Edinburgh, UK

Running title: The trafficking protein Ambra1 regulates transcription

*Corresponding author: Prof Margaret Frame CRUK Edinburgh Centre Institute of Genetics and Molecular Medicine University of Edinburgh Crewe Road South EH4 2XR Edinburgh UK

Keywords: Ambra1, trafficking, , nucleus, transcription, Atf2, chromatin, Akap8, Cdk9

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The trafficking protein Ambra1 regulates transcription

suppresses its binding to the E3-ligase ABSTRACT TRAF1 and the ubiquitylation of ULK1, Previously, we reported that Ambra1 is a thereby controlling the stability and core component of a cytoplasmic function of ULK1 (8). During apoptosis, trafficking network, acting as a spatial caspases and calpains mediate cleavage as rheostat to control active Src and FAK well as degradation of Ambra1 (9). levels in addition to its critical roles in Furthermore, Ambra1 expression is autophagy during neurogenesis. Here we regulated by RNF2-dependent identify a novel nuclear scaffolding ubiquitylation, resulting in degradation function for Ambra1 that controls (10). Ambra1 is also involved in the expression. Ambra1 binds to nuclear pore regulation of mitophagy (11). proteins, to other adaptor proteins like Ambra1 has been both positively and FAK and Akap8 in the nucleus, as well as negatively implicated in cancer. Thus far, to chromatin modifiers and transcriptional it has been proposed as a tumour regulators such as Brg1, Cdk9 and the suppressor, supporting the binding of c- cAMP-regulated transcription factor Atf2. Myc to the phosphatase PP2A, resulting in Ambra1 contributes to their association c-Myc degradation as well as reduced with chromatin and we identified proliferation and tumourigenesis (12). whose transcription is regulated by Ambra1 has been positively implicated in Ambra1 complexes, likely via histone cholangiocarcinoma, where modifications and phospho-Atf2- overexpression is correlated with invasion dependent transcription. Therefore, and poor survival (5). In addition, through Ambra1 scaffolds protein complexes at its ability to bind PP2A, Ambra1 stabilises chromatin, regulating transcriptional the transcription factor FOXO3, triggering signalling in the nucleus; in particular, it FOXP3-mediated transcription, and T-cell recruits chromatin modifiers and differentiation and homeostasis (13). transcriptional regulators to control expression of genes such as angpt1, tgfb2, We reported previously that in squamous tgfb3, itga8 and itgb7 that likely contribute cell carcinoma (SCC) cells derived from to the role of Ambra1 in cancer cell the mutated oncogenic H-Ras driven invasion. DMBA/TPA model of carcinogenesis, Ambra1 is a Focal Adhesion Kinase

(FAK)- and Src-binding partner, INTRODUCTION regulating cancer-related phenotypes like cancer cell polarisation and chemotactic Ambra1 (Activating Molecule in Beclin1- Regulated Autophagy) is already known to invasion (4,14). In FAK-depleted SCC be an important protein in physiology, e.g. cells, Ambra1 is involved in the targeting of active Src to intracellular autophagic in the development of the central nervous system, vertebrate embryogenesis, adult puncta, while an Ambra1-binding neurogenesis and cancer cell invasion (1- impaired FAK mutant retains more active FAK and Src at focal adhesions, resulting 5). However, our understanding of the full range of functions of this crucial cellular in increased cell adhesion and invasion. regulator is unknown. As an important We concluded that Ambra1 lies at the autophagy regulator, Ambra1 binds heart of an intracellular trafficking network in SCC cells, regulating the Beclin1 and is involved in the initiation of autophagy that is needed for neurogenesis localisation of active FAK and Src (2). In the absence of autophagy, the required for cancer processes (4). Ambra1/Beclin1/Vps34 complex is bound Here, we investigate the nuclear function to the dynein motor complex; when of Ambra1, and show that it binds to FAK autophagy is induced, the kinase ULK1 in the nucleus, as well as to other nuclear phosphorylates Ambra1, resulting in its adaptor proteins, nuclear pore release from the dynein complex (6,7). components, histone modifying enzymes Additionally, the function of Ambra1 is and regulators of transcription – in some negatively regulated by mTOR that cases regulating their recruitment to

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The trafficking protein Ambra1 regulates transcription

chromatin. Specifically, Ambra1 forms at higher levels in the perinuclear fraction. complexes with Akap8, Brg1 as well as Fraction purities were confirmed by Atf2 and is responsible for the recruitment blotting respective fractions with anti- of Akap8, Bgr1, the Mediator complex GM130, anti-PDI, anti-Lamin A/C and components Cdk9 as well as p-Atf2 T71 to anti-GAPDH (Supplementary Figure 1B). chromatin. Both Ambra1 and its binding Further, in contrast to FAK, Ambra1 can scaffold protein Akap8 regulate the also be detected in nuclear extracts from binding of transcriptional proteins to primary mouse keratinocytes chromatin, especially p-Atf2 T71, and (Supplementary Figure 1C) (16). modulate histone modifications. The Nuclear Ambra1 binds proteins binding of Atf2/p-Atf2 T71 to chromatin involved in transcription is most likely regulated by the Ambra1- interacting proteins Cdk9. Therefore, Next, we investigated the nature of protein Ambra1 acts as a scaffold protein in the binding partners of Ambra1 in the nucleus. nucleus, recruiting regulators of Highly purified nuclear extracts of SCC transcription to chromatin. This creates an FAK-WT and -/- cells were obtained by Ambra1-dependent nuclear microdomain sucrose gradient centrifugation and used that regulates . for Ambra1 immunoprecipitations (anti- rabbit IgG served as a negative control),

and specific nuclear binding proteins were RESULTS determined by quantitative label-free mass spectrometry (see Materials and Methods). Ambra1 localises to the nucleus Amongst the nuclear Ambra1 interacting Here we show that Ambra1 not only proteins identified were several proteins locates at focal adhesions and in that form part of the nuclear pore complex intracellular autophagic puncta in SCC (functional interaction network shown in cells, but also in the nucleus (Figure 1A). Figure 1C). Indeed, we had previously Staining with only secondary antibodies observed that the nuclear pore protein Tpr (anti-rabbit 488 and anti-mouse 594) ruled was an Ambra1-interacting protein using out unspecific false-positive nuclear whole cell lysates (4) – together implying staining (Supplementary Figure 1A). In that Ambra1 was associated with nuclear order to confirm Ambra1 was nuclear pore proteins either during entry into the biochemically, fractions of SCC FAK-WT nucleus or as part of its nuclear functions. and -/- were isolated and subjected to analysis for biological Western blotting (Figure 1B). Biochemical processes of proteins that bind Ambra1 in nuclear isolations were checked by nuclear fractions revealed transcription, blotting with anti-GAPDH, anti-Lamin RNA/mRNA processing, histone A/C and anti-H4. In the nuclear fractions modification and chromatin modification of these SCC cells, we could detect as the most highly represented categories Ambra1 as well as FAK, the latter in line (Supplementary Figure 2). As a result of with our previous reports (15,16). The these analyses, we further examined nuclear localisation of Ambra1 was not nuclear Ambra1-interactors involved in dependent on FAK, as nuclear Ambra1 is the regulation of transcription and used present at indistinguishable levels in these hits to build a protein interaction nuclear fractions from both FAK-deficient network based on known direct physical (FAK -/-) SCC cells and the same cells re- interactions (Figure 1D). Amongst these expressing wild type FAK to similar levels were several components of the Mediator as parental SCC cells (FAK-WT) (17,18). complex, a multiprotein complex In more highly purified cellular fractions functioning as a transcriptional coactivator of SCC FAK-WT and -/- cell lysates, for RNA Polymerase II (highlighted in extracting cytosolic (C), perinuclear (PN) red) (19,20). Also present were several and nuclear (N) fractions (Supplementary components of the SWI/SNF Methods, Supplementary Figure 1B), (SWItch/Sucrose Non-Fermentable) Ambra1 was present at comparable levels nucleosome remodelling complex that in the cytosolic and nuclear fractions, and allows transcription factor binding by

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The trafficking protein Ambra1 regulates transcription

‘opening up’ chromatin structure, e.g. the Since Ambra1 has predominantly been catalytic subunit SMARCA4 (Brg1), defined as a scaffold protein involved in which allows ATP dependent chromatin intracellular trafficking/autophagy thus remodelling (highlighted in blue) (21-23). far, we hypothesized that Ambra1 might Nuclear Ambra1 was also found to interact also serve as a scaffold protein in the with several members of the cAMP nucleus. Therefore, we investigated dependent AP-1 complex, including c-Jun, whether reducing Ambra1 levels using Fosl, Atf2 (a member of the CREB (cAMP efficient siRNA pool-mediated depletion response element binding) family of that we have used previously (4) did not leucine zipper proteins) and Atf7, which significantly alter the levels of its also binds to nuclear FAK (highlighted in interacting proteins in whole cell lysates or green; (16,24). Since cAMP-regulated nuclear extracts (Figure 3A). We next transcription factors were amongst nuclear isolated chromatin from SCC FAK-WT Ambra1-binding proteins, we also noted cells after Ambra1 depletion and probed that nuclear Ambra1 binds to Akap8 (A for FAK, Brg1, Akap8, Atf2 and its kinase anchor protein 8, also known as phosphorylated (and activated) form p- Akap95), a scaffold that targets PKA to Atf2 T71 (Figure 3B, C). Reduced cAMP-responsive elements in the nucleus, expression of Ambra1 suppressed the and which is linked to chromatin status binding of FAK, Brg1, Akap8 and p-Atf2 and retention of p90 S6K to the nucleus (but not visibly total Atf2) to chromatin to (25-28). Interestingly, both Atf2 and a greater or lesser extent. This suggests Akap8 were also identified by proteomics that, as well as Ambra1-mediated as Ambra1 binding proteins using whole chromatin recruitment, there are likely cell lysates in our previous experiments other routes by which binding partners are (4). recruited to chromatin since their recruitment is reduced, but not diminished, We next selected a number of proteins upon Ambra1 loss. In these analyses, we identified by mass spectrometry analyses, also included the Cdk8 and Cdk9, i.e. Nup153, Akap8, Brg1, Atf2, and the components of the Mediator complex that RNA polymerase II Rpb1 (all highlighted modulates RNA polymerase II-mediated with red circles in Figure 1C and D) and transcription (29-31). These were included confirmed their binding with Ambra1 in because they were: (1) identified as the nucleus by co-immunoprecipitation in binding to Ambra1 in mass spectrometry nuclear fractions (Figure 2A – E). One experiments (Figure 1D, highlighted with question we had, given our previously a red circles), and (2) are predicted to be reported co-functioning of Ambra1 and enzymes with the potential to FAK in the cytoplasm, was whether or not phosphorylate Atf2 at T71 residue on FAK may be regulating the nuclear chromatin that we observe in the presence translocation of its binding partners, such of Ambra1 (Figure 3; predictions via the as Ambra1 that also locates to the nucleus. Biocuckoo phosphorylation prediction We did not find any significant difference site: Cdk8 (score 40.329; cut-off 29.727), in the nuclear levels of Ambra1 or its Cdk9 (score 7.393; cut-off 2.931). In interaction with the nuclear binding keeping with other Ambra1-binding partners examined between SCC cells proteins studied here, we found that Cdk9, expressing FAK-WT when compared to but not Cdk8, binding to chromatin was FAK-deficient (-/-) cells; we therefore reduced upon depletion of Ambra1 (Figure conclude that FAK does not regulate the 3B, C). In this regard, we note that Cdk9 trafficking of Ambra1 to the nucleus or has been reported to bind another Ambra1 associations there. Therefore, in SWI/SNF complex component, future experiments, we have generally SMARCB1, which has also been only presented data from SCC cells identified as a nuclear Ambra1 binding expressing FAK-WT. protein (32), implying there are likely Loss of Ambra1 causes reduced other components of Ambra1 complexes association of interacting partners with that link to chromatin remodelling. Taken chromatin together, our data imply that Ambra1

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The trafficking protein Ambra1 regulates transcription

forms complexes in the nucleus with other and also Atf2 phosphorylation/activation. protein scaffolds, such as the PKA Thus, Ambra1 and Akap8, which form a scaffold Akap8, chromatin modifiers and complex in the nucleus of SCC cells, both transcription factors, including Atf2. contribute to the recruitment of active Atf2 Akap8 also regulates the level of p-Atf2 (p-Atf2 T71) to chromatin, likely via the at chromatin Mediator complex component Cdk9 downstream (see model in Figure 4E). Ambra1 binds the PKA scaffold Akap8 Therefore, an obvious question that and is required for its optimal binding to follows is whether Ambra1, Akap8 and chromatin. Upon depletion of Akap8 by Atf2 co-regulate the expression of a sub- pooled siRNA, we found that whilst set of genes. neither Ambra1 (nor FAK) binding to chromatin was affected, showing it was Ambra1, Akap8, CDK9 and Atf2 co- downstream of Ambra1, the level of p- regulate a sub-set of genes Atf2 T71 associated with chromatin was The data presented to this point showed reduced (Figure 4A, B). This implies a that Ambra1 localises to the nucleus, model (Figure 4E) whereby Ambra1 is associates with chromatin and interacts upstream of recruitment of Akap8 to with nuclear proteins that regulate chromatin, and Akap8, in turn, is required transcription. Both Ambra1 and its binding for optimal chromatin association of active partner Akap8 recruit transcription factors, p-Atf2 that is also Ambra1-dependent. such as Atf2; the latter is proposed to Total Atf2 recruitment was not reduced by result in histone modifications and altered depletion of Akap8, suggesting that a chromatin accessibility, leading to specific function of Akap8 may be to transcriptional changes (40). To address recruit the enzyme that phosphorylates whether there were genes whose Atf2 at chromatin. We noted that the transcription was co-regulated by Ambra1, activity of Atf2 is proposed to be regulated Akap8 and Atf2, SCC FAK-WT cells were by phosphorylation at several residues, transfected with siControl, siAmbra1, including T71, by kinases such as ERK, siAkap8 or siAtf2 siRNA. A sub-set of JNK, p38 and PLK3, promoting Atf2 genes whose expression was changed by heterodimerisation and increased all three depletions was identified using transcription and histone acetyl transferase the Nanostring PanCancer Mouse (HAT) activity (24,33-39). However, Pathways Panel. In total we identified 94 using inhibitors of the kinases proposed genes that were significantly (p < 0.05) at above to phosphorylate Atf2 on T71, we least 2-fold up- or downregulated did not find evidence for a role for any of compared to control siRNA (Figure 5A, B; these in regulating Atf2 phosphorylation at Supplementary Figure 3A – F). Ambra1, chromatin in SCC cells used here (not Atf2 or Akap8 depletion significantly shown). Therefore, we examined the altered the expression of 18 genes from Mediator complex kinases Cdk8 and this panel (Figure 5A, B). In order to Cdk9, which, as mentioned previously, validate the gene expression changes, we may potentially phosphorylate Atf2 at performed qRT-PCR for the co-regulated T71. We therefore depleted Cdk8 and genes angpt1, tgfb2, tgfb3, itga8 and itgb7 Cdk9 in SCC FAK-WT cells and prepared Figure 5d, e). For angpt1, tgfb2, tgfb3 and nuclear and chromatin fractions to probe itga8 we confirmed the downregulation for p-Atf2 T71. We found that depletion of upon siRNA transfection (Figure 5E), Cdk9 resulted in reduced chromatin- whilst upregulation of itgb7 was also associated p-Atf2 T71 (Figure 4C, D) that confirmed (Figure 5E). In addition, KEGG is also Ambra1 and Akap8-dependent; (Kyoto Encyclopedia of Genes and however, in this case total Atf2 recruited Genomes) analysis of Ambra1, Akap8 and to chromatin was also reduced. These Atf2 regulated genes revealed the top findings imply that the Mediator complex enriched signalling pathway gene sets component, Cdk9, which binds to Ambra1 ‘PI3K-Akt signalling pathway’, ‘pathways in the nucleus, controls recruitment of in cancer’, ‘focal adhesion’ and ‘MAPK Atf2 to chromatin downstream of Ambra1

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The trafficking protein Ambra1 regulates transcription

signalling pathway’ were in common and SET1 (Figure 6A). Further, gene (Figure 5C; for full list see Supplementary ontology analysis for biological processes Figure 4), strongly suggesting an overlap of nuclear Ambra1 interactors identified in the functions of the genes regulated by covalent chromatin and histone Ambra1, Akap8 and Atf2 complexes. modifications as well as chromatin remodelling as categories (Supplementary Since we found the nuclear Ambra1- Figure 2). In addition, Akap8 has been binding Mediator complex protein Cdk9 to reported to bind to Dpy30, a core subunit be part of the regulation of p-Atf2 at of H3K4 histone methyltransferases (41). chromatin in SCC cells, (Figure 4C, D), Therefore, we next addressed whether we next addressed whether Cdk9 was also Ambra1 and Akap8 influence histone implicated in the transcriptional regulation modifications by transfecting SCC FAK- of the above co-regulated genes. Depletion WT and -/- cells with siControl, siAmbra1 of Cdk9 by siRNA resulted in broadly (Figure 6B, C) or siAkap8 (Figure 6D, E) similar gene expression changes to that and examined the histone modifications observed upon depletion of Ambra1, H3K4me2, H3Kme3 as well as H3K27Ac Akap8 or Atf2, e.g. expression of angpt1, by Western blotting. Tri-methylation of tgfb2, tgfb3 and itga8 was reduced (Figure Lysine 4 and acetylation of Lysine 27 on 5F), whilst itgb7 was increased in histone 3 are generally regarded as qualitative agreement with the effects of positive indicators of transcriptional depleting Ambra1, Akap8 or Atf2 (Figure activation, promoting gene expression (42- 5F). 45). Depletion of Ambra1 or Akap8 Taken together, these results indicate that reduced both di- and tri-methylation of Ambra1, Akap8, Cdk9 and Atf2 are H3K4 as well as acetylation of H3K27 complexed together and are at chromatin, (irrespective of whether FAK was present likely at the promoters of co-regulated or not). This indicates that both Ambra1 genes described above, and potentially and Akap8 can influence histone many more not represented in the modifications, likely as a result of Nanostring PanCancer Mouse Pathways interacting histone modifying enzymes, Panel used here. The presence of which could result in chromatin chromatin modifiers in the nuclear remodelling and altered accessibility of Ambra1 interactome, validated in the case transcription factors. of Brg1 (Figure 1 and 2C), suggested the

intriguing possibility that the novel transcriptional regulatory pathway we DISCUSSION describe is, at least in part, regulated by Here we describe a completely novel chromatin accessibility. In keeping with transcriptional signalling pathway this, the functioning of Atf2 that is controlled by the scaffold protein Ambra1 phosphorylated at T71 downstream of in the nucleus. Like Ambra1, other Ambra1 and Akap8, is known to promote proteins involved in autophagy have been HAT activity and transcription (35,38). reported in the nucleus, e.g. LC3B binds to Ambra1 and Akap8 regulate histone Lamin B1, mediating the degradation of modifications the nuclear lamina and Beclin 1, promoting autophagy-independent DNA Functional interaction network analysis of damage repair (46,47). No typical nuclear nuclear Ambra1 binding partners localisation sequence is evident for identified by mass spectrometry revealed Ambra1, and hence the mechanism of several components of histone nuclear translocation is unknown; modification complexes, including histone however, as nuclear Ambra1 interacts with acetylation complexes NSL (Non-specific components of nuclear pore complexes lethal complex), NuA4 (nucleosome and importins (Figure 1C), it is perhaps acetyltransferase of H4) and PCAF likely that nuclear import of Ambra1 (p300/CBP-associated factor), as well as occurs via binding to these in some way. histone methylation complexes Nuclear Ambra1 interacts with chromatin MLL1/MLL (mixed-lineage leukemia 1)

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The trafficking protein Ambra1 regulates transcription

modifiers and transcriptional regulators in genes that were altered after depletion of the nucleus, including those also identified Ambra1, Akap8 and Atf2, we found that as proteins that bind to FAK and IL33, e.g. (according to SMARCC1, Ruvbl1 and Ruvbl2 (15), www.natural.salk.edu/CREB) tgfb2 and suggesting there may be a link between tgfb3 both have CRE_TATA boxes that Ambra1 and FAK functions in the nucleus might serve as Atf2 binding sites. Future as well as in the cytoplasm (4). In this work will examine whether Ambra1 regard, we did find that depletion of influences the binding of p-Atf2 T71 at the Ambra1 leads to reduced FAK recruitment promoters of these genes. Moreover, it is to chromatin. likely that some of the cancer-associated functions of Ambra1 in SCC cells (such as The PKA-scaffold Akap8 that binds to cancer cell invasion) are associated with Ambra1 in the nucleus has itself the nuclear transcription signalling effects previously been linked to histone we report here, e.g. on TGFβ isoforms as modifications and chromatin changes. well as its trafficking effects we reported Indeed, by interacting with the previously (4). MLL1/MLL complex via Dpy30, Akap8 regulates histone H3K4 methyltransferase Taken together, our data lead us to complexes and binds to the nuclear matrix, propose the following model (depicted in nucleoporin component Tpr as well as Figure 7); briefly, Ambra1 was already chromatin; in turn, this contributes to known to localise to autophagosomes in condensation and the cytoplasm and to focal adhesions, transcription, effects that are important for where it regulates the removal of the mitotic checkpoint (41,48-51). Akap8 ‘untethered’ tyrosine kinases via also binds the histone deacetylase HDAC3 autophagy (2,4). We now show that and influences mitosis (52). Therefore, Ambra1 also interacts with nuclear pore existing studies had already suggested a proteins, and locates to the nucleus where scaffolding function for Akap8 in it is part of a network of inter-linked assembly of chromatin modification chromatin modifiers and transcriptional complexes. Akap8 dissociates from regulators, including a set of interacting chromatin and the nuclear matrix as a proteins whose recruitment to chromatin is result of nuclear tyrosine phosphorylation, influenced by Ambra1. This includes the and it may have a role in regulation of PKA-scaffold Akap8, the Mediator chromatin structural changes (53). Finally, complex component Cdk9 and the a recent study confirmed the scaffold transcription factor Atf2 in its active form. function of Akap8 in organising nuclear Moreover, Ambra1, Akap8, Cdk9 and microdomains, thereby controlling local Atf2 co-regulate the expression of a sub- cAMP for nuclear PKA regulation (54) – set of genes. Both Ambra1 and Akap8 although it is not clear whether this is a influence cellular histone modification that chromatin-associated function of Akap8. could contribute to their transcriptional effects. Therefore, we have uncovered a Nuclear envelope and nuclear pore completely novel function for the components, like Nup153, associate with autophagy protein Ambra1, which acts as chromatin and regulate genome a nuclear scaffold to recruit other scaffold organisation and gene expression via proteins, chromatin modifiers and nuclear pore complexes acting as scaffold transcriptional regulators to elicit gene platforms to allow the assembly and expression changes via Atf2. A similar recruitment of transcription factors to the scaffolding mechanism creating nuclear nuclear periphery (55,56). Therefore, ‘transcription signalling hubs’ has been Ambra1 might serve as a molecular described for FAK, controlling ccl5, IL33, scaffold that links chromatin to the nuclear tgfb2 and igfbp3 transcription as well as pore complex, allowing transcription for mAKAPβ, which creates nuclear factor binding (such as Atf2) and resulting ‘signalosomes’ and binds the gene expression. When we looked at transcriptional regulators NFAT, MEF2 potential Atf2-binding sequences in the and HIF1α (16,57,58).

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The trafficking protein Ambra1 regulates transcription

MATERIALS AND METHODS City, UK). Lysates were incubated on ice Antibodies for 10 minutes and centrifuged. The Antibodies used were as follows: anti- resulting pellets were washed twice in Paxillin and anti-GM130 antibodies (BD DET buffer, resuspended in RIPA buffer Transduction Laboratories, New Jersey, and cleared by centrifugation. USA), anti-Akap8 and anti-Nup153 For mass spectrometry, nuclear lysates (Abcam, Cambridge, UK), anti-FAK, anti- were prepared using Nuclei PURE prep PDI, anti-p-Atf2 T71, anti-Atf2, anti-Brg1, isolation kit (Sigma, Gillingham, UK). anti-Rpb1, anti-Cdk8, anti-Cdk9, anti- Chromatin isolation Histone H4, anti-H3K4me2, anti- The protocol was adapted from H3K4me3, anti-H3K27Ac, anti-Histone McAndrew et al., (59). All buffers were H3, anti-Lamin A/C and anti-GAPDH supplemented with PhosStop and (Cell Signaling Technologies, Danvers, Complete Ultra Protease Inhibitor tablets MA, USA), as well as anti-Ambra1 (Roche, Welwyn Garden City, UK). antibody (Millipore, Billerica, MA, USA). Briefly, cells were washed twice in ice- Anti-rabbit or mouse peroxidase- cold PBS, lysed in extraction buffer (10 conjugated secondary antibodies were mM Hepes pH 7.9, 10 mM KCl, 1.5 mM purchased from Cell Signaling MgCl2, 0.34M sucrose, 10% glycerol, Technologies. 0.2% NP-40) and centrifuged for 5 Cell culture minutes at 6500 g. Nuclear pellets were FAK-deficient Squamous Cell Carcinoma washed in extraction buffer without NP-40 (SCC) cell lines were generated as and centrifuged for 5 minutes at 6500 g. described previously (18). SCCs were Pellets were resuspended in Low salt maintained in Glasgow MEM containing buffer (10 mM Hepes pH 7.9, 3 mM 10% FCS, 2 mM L-glutamine, non- EDTA, 0.2 mM EGTA) and incubated for essential amino acids, sodium pyruvate 30 minutes at 4 °C with rotation, before and MEM vitamins at 37°C, 5% CO2. SCC centrifugation for 5 minutes at 6500 g. FAK-WT cells were maintained in 1 Pellets were resuspended in High salt mg/ml hygromycin B. buffer (50 mM Tris-HCl pH 8.0, 2.5 M siRNA NaCl, 0.05% NP-40) and incubated for 30 FAK-WT or FAK -/- SCC cells were minutes at 4 °C with rotation. transiently transfected using HiPerFect Supernatants containing chromatin (Qiagen, Manchester, UK), according to fractions were cleared by centrifugation. manufacturer’s protocol with a final Proteins were precipitated by adding TCA concentration of 80 or 100 nM siRNA to a final volume of 10% and incubating respectively (Supplementary Material and on ice for 15 minutes. Precipitated proteins Methods, Table 1). Cells were analyzed at were pelleted by centrifugation, washed 48 h post transfection. twice with ice-cold acetone and then Whole cell lysates resuspended in 2x sample buffer prior to Cells were washed twice in ice-cold PBS analysis by Western blotting. and then lysed in RIPA buffer (50 mM Immunoblotting, immunoprecipitation Tris-HCl pH 8.0, 150 mM NaCl, 1% and mass spectrometry Triton X-100, 0.1% SDS and 0.5% sodium Protein concentration was calculated using deoxycholate) supplemented with a BCA protein assay kit (Thermo PhosStop and Complete Ultra Protease Scientific, Loughborough, UK). For Inhibitor tablets (Roche, Welwyn Garden immunoprecipitation, 1 mg lysates were City, UK) and cleared by centrifugation. incubated with 2 µg of unconjugated Nuclear fractionation antibodies at 4°C overnight with agitation. Cells were washed twice in ice-cold PBS Unconjugated antibody samples were and then lysed in DET buffer (150 mM incubated with 10 µl of Protein A agarose NaCl, 25 mM Hepes pH 7.5, 1 mM β- for 1 h at 4°C. Beads were washed three Mercaptoethanol, 0.2 mM CaCl2, 0.5 mM times in lysis buffer and once in 0.6 M MgCl2, 0.5% NP-40) supplemented with LiCl, resuspended in 20 µl 2x sample PhosStop and Complete Ultra Protease buffer (130 mM Tris pH 6.8, 20% Inhibitor tablets (Roche, Welwyn Garden glycerol, 5% SDS, 8% β-mercaptoethanol,

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The trafficking protein Ambra1 regulates transcription

bromphenol blue) and heated for 5 Panel (Nanostring, Amersham, UK). minutes at 95°C. Samples were then Analysis was performed using nSolver subjected to SDS-PAGE analysis as analysis software (Nanostring, Amersham, described elsewhere (4). UK). The cut-off point of statistically For mass spectrometry, Ambra1 was significant relative changes immunoprecipitated from 2 mg nuclear (siRNA/siControl p < 0.05) was set to 2- lysates of SCC FAK-WT and -/- cells fold. (samples in triplicates), using 2 µg of Statistical tests unconjugated antibodies (anti-Ambra1 and For all experiments shown, n = 3 – 5. rabbit-anti-IgG) at 4°C overnight with Error bars for the graphs show s.d. agitation. Unconjugated antibody samples Student’s t-test was carried out to calculate were incubated with 20 µl of Protein A the statistical significance. agarose for 1 h at 4°C. Beads were washed

twice in lysis buffer and twice in PBS. Protein complexes were subjected to on- ACKNOWLEDGEMENTS bead proteolytic digestion, followed by This work was funded by CR-UK desalting and liquid chromatography- Programme Grant (C157/A24837) to M tandem mass spectrometry as reported Frame and V Brunton. We would like to previously (60). The interaction network thank Jimi Wills and Alexander von analysis was performed using Cytoscape. Kriegsheim at the IGMM Mass Immunofluorescence microscopy and Spectrometry Facility for the mass image analysis spectrometry analysis and Alison Munro at Cells were fixed, stained and imaged as the IGMM HTPU array facility for the described in Schoenherr et al., (4). Nanostring analysis. qRT-PCR

RNA from cells was isolated using the AUTHOR CONTRIBUTIONS RNeasy Mini Kit (Qiagen, Manchester, C.S. designed and performed the cell UK). 500 ng of total RNA was reverse- biology and biochemical experiments and transcribed using the SuperScript First- co-wrote the paper. M.C.F. conceived Strand cDNA synthesis kit (Life proteomics, provided funding through Technology, Paisley, UK). For the PCR competitively awarded grants and co- amplification in a Step One Plus real-time wrote the paper. PCR system (Life Technology, Paisley,

UK) 25 ng cDNA were used in a total COMPETING INTERESTS reaction mix of 20 µl containing 10 ul STATEMENT Sensi Fast SYBR Green Hi-Rox (Bioline, The authors declare no competing London, UK) as well as 400 nM forward interests. and reverse primer (Supplementary

Material and Methods, Table 2). GAPDH was used to control for differences in FIGURE LEGENDS cDNA input. Relative expression was Figure 1: Nuclear Ambra1 binds calculated according to the ΔΔCt chromatin modifiers and transcriptional quantification method. Each sample within regulators. (A) Representative an experiment was performed in triplicate immunofluorescence images of SCC and the experiment was carried out three FAK-WT and -/- cells which were grown times. on glass coverslips for 24 h, fixed and Nanostring gene expression analysis stained with anti-Ambra1, anti-Paxillin SCC FAK-WT cells were transfected with and DAPI. Scale bars, 20 µm. (B) Whole siControl, siAmbra1, siAkap8 and siAtf2. cell and nuclear lysates of SCC FAK-WT RNA from cells was isolated 48 h post and -/- cells were subjected to Western transfection using the RNease Mini Kit blot analysis with anti-Ambra1 and anti- (Qiagen, Manchester, UK) and diluted to FAK. Anti-GAPDH, anti-Lamin A/C and 20 ng/µl. Samples (in triplicates) were anti-H4 served as controls for the purity of subjected to gene expression analysis the nuclear extracts as well as loading using the PanCancer Mouse Pathways

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The trafficking protein Ambra1 regulates transcription

controls. (C, D) Gene ontology enrichment protein levels normalised to Histone H4. analysis for biological processes of nuclear Figure 4: Depletion of Akap8 and Cdk9 Ambra1-binding proteins forming part of reduce p-Atf2 T71 binding to the nuclear pore complex (C) and being chromatin. (A) SCC FAK-WT cells were involved in transcription (D) in SCC FAK- transfected with siControl and siAkap8 WT and -/- cells. Hits were filtered for (siGENOME pool). After 48 h whole cell statistical significant (p < 0.05) 2-fold lysates and chromatin extracts were enrichment over IgG control and analysed by Western blot using anti- subsequently used to build a protein Ambra1, anti-FAK, anti-p-Atf2 T71, anti- interaction network based on direct Atf2 and anti-Akap8. Anti-Histone H4 physical interactions (grey lines). served as a marker for chromatin as well Components of various complexes as a loading control. (B) The graph shows involved in transcription are highlighted: relative chromatin protein levels SWI/SNF complex (blue), cAMP normalised to Histone H4. (C) SCC FAK- regulated transcription factor (green) and WT cells were transfected with siControl, Mediator complex (red). Ambra1- siCdk8 and siCdk9 (siGENOME pool). interacting proteins selected for further After 48 h whole cell lysates, nuclear and investigation are highlighted with a red chromatin extracts were analysed by circle. Western blot using anti-pAtf2 T71, anti- Figure 2: Nuclear Ambra1 binding to Atf2, anti-Cdk8 and anti-Cdk9. Anti- mass spectrometry-identified Lamin A/C, anti-GAPDH and anti-Histone interaction partners. Ambra1 was H4 served as a control for the purity of immunoprecipitated from whole cell and nuclear and chromatin extracts as well as a nuclear lysates of SCC FAK-WT and -/- loading control. (D) The graph shows cells using anti-Ambra1, followed by relative nuclear or chromatin protein levels Western blot analysis with anti-Ambra1 normalised to Lamin A/C or Histone H4 and anti-Nup153 (A), anti-Akap8 (B), respectively. (E) Hypothesis model. In the anti-Brg1 (C), anti-Atf2 (D) and anti-Rpb1 nucleus of SCC cells Ambra1 and Akap8 (E). Anti-Lamin A/C and anti-GAPDH form a complex and contribute to the were used as a control for the purity of the recruitment of active Atf2 (p-Atf2 T71) to nuclear lysates as well as a loading chromatin, most likely downstream via the control. Mediator complex component Cdk9. This strongly suggests that Ambra1 might be Figure 3: Loss of Ambra1 leads to involved in chromatin remodeling and reduced association of interacting transcription. Further, together with Akap8 proteins with chromatin. (A) SCC FAK- and Atf2 it might co-regulate the WT cells were transfected with siControl expression of a sub-set of genes. and siAmbra1 (siGENOME pool). After 48 h whole cell and nuclear lysates were Figure 5: Ambra1, Akap8, Cdk9 and analysed by Western blot using anti- Atf2 co-regulate a sub-set of genes. SCC Ambra1, anti-FAK, anti-Brg1, anti-Akap8, FAK-WT cells were transfected with anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and siControl, siAmbra1, siAkap8 and siAtf2 anti-Atf2. Anti-Lamin A/C and anti- (siGENOME pool). RNA was isolated 48 GAPDH were used as a control for the h post transfection and subjected to gene purity of the nuclear lysates as well as a expression analysis using the PanCancer loading control. (B) SCC FAK-WT cells Mouse Pathways Panel. (A) Venn diagram were transfected with siControl and of significantly altered genes compared to siAmbra1. After 48 h whole cell lysates siControl (p < 0.05, 2-fold difference and chromatin extracts were analysed by compared to siControl). (B) Relative gene Western blot using anti-Ambra1, anti- expression of 18 co-regulated genes by FAK, anti-Brg1, anti-Akap8, anti-Cdk8, Ambra1, Akap8 and Atf2 knockdown anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. compared to siControl. (C) Table of the Anti-Histone H4 served as a marker for top five enriched signalling pathway gene chromatin as well as a loading control. (C) sets according to KEGG (Kyoto The graph shows relative chromatin Encyclopedia of Genes and Genomes)

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The trafficking protein Ambra1 regulates transcription

pathway analysis of Ambra1, Akap8 and components and is translocated into the Atf2 regulated genes. (D) Western blot nucleus most likely via nuclear pores and showing Atf2 knockdown by siRNA in importins. Nuclear Ambra1 is part of a SCC FAK-WT cells. Anti-GAPDH served network consisting of chromatin modifiers as a loading control. (E, F) Validation of and transcriptional regulators, some of Ambra1, Akap8 and Atf2 mediated which are recruited to chromatin in an angpt1, tgfb2, tgfb3, itga8 and itgb7 Ambra1-mediated manner, including the expression changes by qRT-PCR using PKA-scaffold Akap8, Cdk9 and active RNA isolated 48 h post transfection of Atf2 (p-Atf2 T71). Further, Ambra1, SCC FAK-WT cells with siControl, Akap8, Cdk9 and Atf2 co-regulate the siAmbra1, siAkap8 and siAtf2 (E) as well expression of a sub-set of genes like as siControl and siCdk9 (F). Error bars: angpt1, tgfb2, tgfb3, itga8 and itgb7. Both s.d. (*) p < 0.01. (#) p < 0.05. Ambra1 and Akap8 influence cellular histone modifications, which could Figure 6: Ambra1 or Akap8 depletion contribute to their transcriptional effects. decreases histone modifications. (A) Overall, the autophagy protein Ambra1 Functional interaction network analysis of also acts as a nuclear platform to recruit nuclear Ambra1 binding partners key scaffolds, chromatin modifiers and identified by mass spectrometry were transcriptional regulators to elicit gene filtered for statistical significant (p < 0.05) expression changes via Atf2. 2-fold enrichment over IgG control and components of histone modification complexes were subsequently used to REFERENCES build a protein interaction network based 1. Benato, F., Skobo, T., Gioacchini, on direct physical interaction (grey lines). G., Moro, I., Ciccosanti, F., These complexes include histone Piacentini, M., Fimia, G. M., acetylation complexes (NSL: Non-specific Carnevali, O., and Dalla Valle, L. lethal); NuA4: nucleosome (2013) Ambra1 knockdown in acetyltransferase of H4; PCAF: zebrafish leads to incomplete p300/CBP-associated factor) and histone development due to severe defects methylation complexes (MLL1/MLL: in organogenesis. Autophagy 9, mixed-lineage leukemia 1; SET1) (all 476-495 highlighted by light blue background). (B 2. Fimia, G. M., Stoykova, A., – E) SCC FAK-WT and -/- cells were Romagnoli, A., Giunta, L., Di transfected with siControl and siAmbra1 Bartolomeo, S., Nardacci, R., (B, C) or siControl and siAkap8 Corazzari, M., Fuoco, C., Ucar, respectively (siGENOME pool) (D, E). A., Schwartz, P., Gruss, P., After 48 h whole cell lysates were Piacentini, M., Chowdhury, K., subjected to Western blot analysis using and Cecconi, F. (2007) Ambra1 anti-Ambra1, anti-Akap8, anti-di- regulates autophagy and H3K4me, anti-tri-H3K4me and anti- development of the nervous H3K27Ac. Anti-GAPDH and anti-Histone system. Nature 447, 1121-1125 H3 served as loading controls. (C, E) The 3. Yazdankhah, M., Farioli- graphs show relative histone modification Vecchioli, S., Tonchev, A. B., levels normalised to Histone H3 or protein Stoykova, A., and Cecconi, F. levels normalised to GAPDH upon (2014) The autophagy regulators Ambra1 (C) or Akap8 (E) knockdown. Ambra1 and Beclin 1 are required Figure 7: Model depicting Ambra1- for adult neurogenesis in the brain dependent transcriptional regulation. In subventricular zone. Cell death & mouse SCC cells, Ambra1 is already disease 5, e1403 known to localise to autophagosomes and 4. Schoenherr, C., Byron, A., focal adhesions, where it binds FAK and Sandilands, E., Paliashvili, K., Src and regulates the removal of Baillie, G. S., Garcia-Munoz, A., untethered kinases via autophagy. Ambra1 Valacca, C., Cecconi, F., Serrels, can also interact with nuclear pore B., and Frame, M. C. (2017)

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The trafficking protein Ambra1 regulates transcription

Ambra1 spatially regulates Src 10. Xia, P., Wang, S., Huang, G., Du, activity and Src/FAK-mediated Y., Zhu, P., Li, M., and Fan, Z. cancer cell invasion via trafficking (2014) RNF2 is recruited by networks. Elife 6 WASH to ubiquitinate AMBRA1 5. Nitta, T., Sato, Y., Ren, X. S., leading to downregulation of Harada, K., Sasaki, M., Hirano, S., autophagy. Cell research 24, 943- and Nakanuma, Y. (2014) 958 Autophagy may promote 11. Strappazzon, F., Nazio, F., carcinoma cell invasion and Corrado, M., Cianfanelli, V., correlate with poor prognosis in Romagnoli, A., Fimia, G. M., cholangiocarcinoma. International Campello, S., Nardacci, R., journal of clinical and Piacentini, M., Campanella, M., experimental pathology 7, 4913- and Cecconi, F. (2015) AMBRA1 4921 is able to induce mitophagy via 6. Di Bartolomeo, S., Corazzari, M., LC3 binding, regardless of Nazio, F., Oliverio, S., Lisi, G., PARKIN and p62/SQSTM1. Cell Antonioli, M., Pagliarini, V., death and differentiation 22, 419- Matteoni, S., Fuoco, C., Giunta, 432 L., D'Amelio, M., Nardacci, R., 12. Cianfanelli, V., Fuoco, C., Romagnoli, A., Piacentini, M., Lorente, M., Salazar, M., Cecconi, F., and Fimia, G. M. Quondamatteo, F., Gherardini, P. (2010) The dynamic interaction of F., De Zio, D., Nazio, F., AMBRA1 with the dynein motor Antonioli, M., D'Orazio, M., complex regulates mammalian Skobo, T., Bordi, M., Rohde, M., autophagy. The Journal of cell Dalla Valle, L., Helmer-Citterich, biology 191, 155-168 M., Gretzmeier, C., Dengjel, J., 7. Strappazzon, F., Vietri-Rudan, M., Fimia, G. M., Piacentini, M., Di Campello, S., Nazio, F., Bartolomeo, S., Velasco, G., and Florenzano, F., Fimia, G. M., Cecconi, F. (2015) AMBRA1 Piacentini, M., Levine, B., and links autophagy to cell Cecconi, F. (2011) Mitochondrial proliferation and tumorigenesis by BCL-2 inhibits AMBRA1-induced promoting c-Myc autophagy. The EMBO journal 30, dephosphorylation and 1195-1208 degradation. Nature cell biology 8. Nazio, F., Strappazzon, F., 17, 20-30 Antonioli, M., Bielli, P., 13. Becher, J., Simula, L., Volpe, E., Cianfanelli, V., Bordi, M., Procaccini, C., La Rocca, C., Gretzmeier, C., Dengjel, J., D'Acunzo, P., Cianfanelli, V., Piacentini, M., Fimia, G. M., and Strappazzon, F., Caruana, I., Cecconi, F. (2013) mTOR inhibits Nazio, F., Weber, G., Gigantino, autophagy by controlling ULK1 V., Botti, G., Ciccosanti, F., ubiquitylation, self-association Borsellino, G., Campello, S., and function through AMBRA1 Mandolesi, G., De Bardi, M., and TRAF6. Nature cell biology Fimia, G. M., D'Amelio, M., 15, 406-416 Ruffini, F., Furlan, R., Centonze, 9. Pagliarini, V., Wirawan, E., D., Martino, G., Braghetta, P., Romagnoli, A., Ciccosanti, F., Chrisam, M., Bonaldo, P., Lisi, G., Lippens, S., Cecconi, F., Matarese, G., Locatelli, F., Fimia, G. M., Vandenabeele, P., Battistini, L., and Cecconi, F. Corazzari, M., and Piacentini, M. (2018) AMBRA1 Controls (2012) Proteolysis of Ambra1 Regulatory T-Cell Differentiation during apoptosis has a role in the and Homeostasis Upstream of the inhibition of the autophagic pro- FOXO3-FOXP3 Axis. survival response. Cell death and Developmental cell 47, 592-607 differentiation 19, 1495-1504 e596

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The trafficking protein Ambra1 regulates transcription

14. Quintanilla, M., Brown, K., 21. Kwon, H., Imbalzano, A. N., Ramsden, M., and Balmain, A. Khavari, P. A., Kingston, R. E., (1986) Carcinogen-specific and Green, M. R. (1994) mutation and amplification of Ha- Nucleosome disruption and ras during mouse skin enhancement of activator binding carcinogenesis. Nature 322, 78-80 by a human SW1/SNF complex. 15. Serrels, B., McGivern, N., Canel, Nature 370, 477-481 M., Byron, A., Johnson, S. C., 22. Phelan, M. L., Sif, S., Narlikar, G. McSorley, H. J., Quinn, N., J., and Kingston, R. E. (1999) Taggart, D., Von Kreigsheim, A., Reconstitution of a core chromatin Anderton, S. M., Serrels, A., and remodeling complex from Frame, M. C. (2017) IL-33 and SWI/SNF subunits. Mol Cell 3, ST2 mediate FAK-dependent 247-253 antitumor immune evasion 23. Malik, S., and Roeder, R. G. through transcriptional networks. (2010) The metazoan Mediator Sci Signal 10 co-activator complex as an 16. Serrels, A., Lund, T., Serrels, B., integrative hub for transcriptional Byron, A., McPherson, R. C., von regulation. Nat Rev Genet 11, 761- Kriegsheim, A., Gomez- 772 Cuadrado, L., Canel, M., Muir, 24. Abdel-Hafiz, H. A., Heasley, L. M., Ring, J. E., Maniati, E., Sims, E., Kyriakis, J. M., Avruch, J., A. H., Pachter, J. A., Brunton, V. Kroll, D. J., Johnson, G. L., and G., Gilbert, N., Anderton, S. M., Hoeffler, J. P. (1992) Activating Nibbs, R. J., and Frame, M. C. transcription factor-2 DNA- (2015) Nuclear FAK controls binding activity is stimulated by chemokine transcription, Tregs, phosphorylation catalyzed by p42 and evasion of anti-tumor and p54 microtubule-associated immunity. Cell 163, 160-173 protein kinases. Mol Endocrinol 6, 17. Sandilands, E., Serrels, B., 2079-2089 Wilkinson, S., and Frame, M. C. 25. Lu, J., Wang, W., Mi, Y., Zhang, (2012) Src-dependent autophagic C., Ying, H., Wang, L., Wang, Y., degradation of Ret in FAK- Myatt, L., and Sun, K. (2017) signalling-defective cancer cells. AKAP95-mediated nuclear EMBO Rep 13, 733-740 anchoring of PKA mediates 18. Serrels, B., Sandilands, E., Serrels, cortisol-induced PTGS2 A., Baillie, G., Houslay, M. D., expression in human amnion Brunton, V. G., Canel, M., fibroblasts. Sci Signal 10 Machesky, L. M., Anderson, K. I., 26. Eide, T., Coghlan, V., Orstavik, and Frame, M. C. (2010) A S., Holsve, C., Solberg, R., complex between FAK, RACK1, Skalhegg, B. S., Lamb, N. J., and PDE4D5 controls spreading Langeberg, L., Fernandez, A., initiation and cancer cell polarity. Scott, J. D., Jahnsen, T., and Curr Biol 20, 1086-1092 Tasken, K. (1998) Molecular 19. Kelleher, R. J., 3rd, Flanagan, P. cloning, chromosomal M., and Kornberg, R. D. (1990) A localization, and cell cycle- novel mediator between activator dependent subcellular distribution proteins and the RNA polymerase of the A-kinase anchoring protein, II transcription apparatus. Cell 61, AKAP95. Exp Cell Res 238, 305- 1209-1215 316 20. Flanagan, P. M., Kelleher, R. J., 27. Collas, P., Le Guellec, K., and 3rd, Sayre, M. H., Tschochner, H., Tasken, K. (1999) The A-kinase- and Kornberg, R. D. (1991) A anchoring protein AKAP95 is a mediator required for activation of multivalent protein with a key role RNA polymerase II transcription in chromatin condensation at in vitro. Nature 350, 436-438

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The trafficking protein Ambra1 regulates transcription

mitosis. The Journal of cell 35. Livingstone, C., Patel, G., and biology 147, 1167-1180 Jones, N. (1995) ATF-2 contains a 28. Gao, X., Chaturvedi, D., and phosphorylation-dependent Patel, T. B. (2012) Localization transcriptional activation domain. and retention of p90 ribosomal S6 The EMBO journal 14, 1785-1797 kinase 1 in the nucleus: 36. Ouwens, D. M., de Ruiter, N. D., implications for its function. Mol van der Zon, G. C., Carter, A. P., Biol Cell 23, 503-515 Schouten, J., van der Burgt, C., 29. Knuesel, M. T., Meyer, K. D., Kooistra, K., Bos, J. L., Maassen, Donner, A. J., Espinosa, J. M., and J. A., and van Dam, H. (2002) Taatjes, D. J. (2009) The human Growth factors can activate ATF2 CDK8 subcomplex is a histone via a two-step mechanism: kinase that requires Med12 for phosphorylation of Thr71 through activity and can function the Ras-MEK-ERK pathway and independently of mediator. Mol of Thr69 through RalGDS-Src- Cell Biol 29, 650-661 p38. The EMBO journal 21, 3782- 30. Knuesel, M. T., Meyer, K. D., 3793 Bernecky, C., and Taatjes, D. J. 37. Song, B., Xie, B., Wang, C., and (2009) The human CDK8 Li, M. (2011) Caspase-3 is a target subcomplex is a molecular switch gene of c-Jun:ATF2 heterodimers that controls Mediator coactivator during apoptosis induced by function. Genes Dev 23, 439-451 activity deprivation in cerebellar 31. De Falco, G., and Giordano, A. granule neurons. Neurosci Lett (2002) CDK9: from basal 505, 76-81 transcription to cancer and AIDS. 38. Kawasaki, H., Schiltz, L., Chiu, Cancer biology & therapy 1, 342- R., Itakura, K., Taira, K., 347 Nakatani, Y., and Yokoyama, K. 32. Shen, J. P., Zhao, D., Sasik, R., K. (2000) ATF-2 has intrinsic Luebeck, J., Birmingham, A., histone acetyltransferase activity Bojorquez-Gomez, A., Licon, K., which is modulated by Klepper, K., Pekin, D., Beckett, A. phosphorylation. Nature 405, 195- N., Sanchez, K. S., Thomas, A., 200 Kuo, C. C., Du, D., Roguev, A., 39. Valdez, B. C., Zander, A. R., Lewis, N. E., Chang, A. N., Song, G., Murray, D., Nieto, Y., Kreisberg, J. F., Krogan, N., Qi, Li, Y., Champlin, R. E., and L., Ideker, T., and Mali, P. (2017) Andersson, B. S. (2014) Combinatorial CRISPR-Cas9 Synergistic cytotoxicity of screens for de novo mapping of gemcitabine, clofarabine and genetic interactions. Nat Methods edelfosine in lymphoma cell lines. 14, 573-576 Blood Cancer J 4, e171 33. Gupta, S., Campbell, D., Derijard, 40. Roth, S. Y., Denu, J. M., and B., and Davis, R. J. (1995) Allis, C. D. (2001) Histone Transcription factor ATF2 acetyltransferases. Annu Rev regulation by the JNK signal Biochem 70, 81-120 transduction pathway. Science 41. Bieluszewska, A., Weglewska, 267, 389-393 M., Bieluszewski, T., Lesniewicz, 34. van Dam, H., Wilhelm, D., Herr, K., and Poreba, E. (2018) PKA- I., Steffen, A., Herrlich, P., and binding domain of AKAP8 is Angel, P. (1995) ATF-2 is essential for direct interaction with preferentially activated by stress- DPY30 protein. FEBS J 285, 947- activated protein kinases to 964 mediate c-jun induction in 42. Santos-Rosa, H., Schneider, R., response to genotoxic agents. The Bannister, A. J., Sherriff, J., EMBO journal 14, 1798-1811 Bernstein, B. E., Emre, N. C., Schreiber, S. L., Mellor, J., and

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The trafficking protein Ambra1 regulates transcription

Kouzarides, T. (2002) Active repair independent of autophagy. genes are tri-methylated at K4 of Sci Rep 7, 45385 histone H3. Nature 419, 407-411 48. Lopez-Soop, G., Ronningen, T., 43. Liang, G., Lin, J. C., Wei, V., Rogala, A., Richartz, N., Yoo, C., Cheng, J. C., Nguyen, C. Blomhoff, H. K., Thiede, B., T., Weisenberger, D. J., Egger, G., Collas, P., and Kuntziger, T. Takai, D., Gonzales, F. A., and (2017) AKAP95 interacts with Jones, P. A. (2004) Distinct nucleoporin TPR in mitosis and is localization of histone H3 important for the spindle assembly acetylation and H3-K4 checkpoint. Cell Cycle 16, 947- methylation to the transcription 956 start sites in the . 49. Akileswaran, L., Taraska, J. W., Proceedings of the National Sayer, J. A., Gettemy, J. M., and Academy of Sciences of the United Coghlan, V. M. (2001) A-kinase- States of America 101, 7357-7362 anchoring protein AKAP95 is 44. Tie, F., Banerjee, R., Stratton, C. targeted to the nuclear matrix and A., Prasad-Sinha, J., Stepanik, V., associates with p68 RNA helicase. Zlobin, A., Diaz, M. O., Scacheri, J Biol Chem 276, 17448-17454 P. C., and Harte, P. J. (2009) CBP- 50. Eide, T., Carlson, C., Tasken, K. mediated acetylation of histone A., Hirano, T., Tasken, K., and H3 lysine 27 antagonizes Collas, P. (2002) Distinct but Drosophila Polycomb silencing. overlapping domains of AKAP95 Development 136, 3131-3141 are implicated in chromosome 45. Creyghton, M. P., Cheng, A. W., condensation and condensin Welstead, G. G., Kooistra, T., targeting. EMBO Rep 3, 426-432 Carey, B. W., Steine, E. J., Hanna, 51. Jiang, H., Lu, X., Shimada, M., J., Lodato, M. A., Frampton, G. Dou, Y., Tang, Z., and Roeder, R. M., Sharp, P. A., Boyer, L. A., G. (2013) Regulation of Young, R. A., and Jaenisch, R. transcription by the MLL2 (2010) Histone H3K27ac complex and MLL complex- separates active from poised associated AKAP95. Nat Struct enhancers and predicts Mol Biol 20, 1156-1163 developmental state. Proceedings 52. Li, Y., Kao, G. D., Garcia, B. A., of the National Academy of Shabanowitz, J., Hunt, D. F., Qin, Sciences of the United States of J., Phelan, C., and Lazar, M. A. America 107, 21931-21936 (2006) A novel histone 46. Dou, Z., Xu, C., Donahue, G., deacetylase pathway regulates Shimi, T., Pan, J. A., Zhu, J., mitosis by modulating Aurora B Ivanov, A., Capell, B. C., Drake, kinase activity. Genes Dev 20, A. M., Shah, P. P., Catanzaro, J. 2566-2579 M., Ricketts, M. D., Lamark, T., 53. Kubota, S., Morii, M., Yuki, R., Adam, S. A., Marmorstein, R., Yamaguchi, N., Yamaguchi, H., Zong, W. X., Johansen, T., Aoyama, K., Kuga, T., Tomonaga, Goldman, R. D., Adams, P. D., T., and Yamaguchi, N. (2015) and Berger, S. L. (2015) Role for Tyrosine Phosphorylation Autophagy mediates degradation of A-kinase Anchoring Protein 8 of nuclear lamina. Nature 527, (AKAP8) in Its Dissociation from 105-109 Chromatin and the Nuclear 47. Xu, F., Fang, Y., Yan, L., Xu, L., Matrix. J Biol Chem 290, 10891- Zhang, S., Cao, Y., Xu, L., Zhang, 10904 X., Xie, J., Jiang, G., Ge, C., An, 54. Clister, T., Greenwald, E. C., N., Zhou, D., Yuan, N., and Baillie, G. S., and Zhang, J. Wang, J. (2017) Nuclear (2019) AKAP95 Organizes a localization of Beclin 1 promotes Nuclear Microdomain to Control radiation-induced DNA damage Local cAMP for Regulating

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The trafficking protein Ambra1 regulates transcription

Nuclear PKA. Cell Chem Biol 26, 58. Dodge-Kafka, K., Gildart, M., 885-891 e884 Tokarski, K., and Kapiloff, M. S. 55. Nanni, S., Re, A., Ripoli, C., (2019) mAKAPbeta signalosomes Gowran, A., Nigro, P., D'Amario, - A nodal regulator of gene D., Amodeo, A., Crea, F., Grassi, transcription associated with C., Pontecorvi, A., Farsetti, A., pathological cardiac remodeling. and Colussi, C. (2016) The Cell Signal 63, 109357 nuclear pore protein Nup153 59. McAndrew, M. J., Gjidoda, A., associates with chromatin and Tagore, M., Miksanek, T., and regulates cardiac gene expression Floer, M. (2016) Chromatin in dystrophic mdx hearts. Remodeler Recruitment during Cardiovasc Res 112, 555-567 Macrophage Differentiation 56. Van de Vosse, D. W., Wan, Y., Facilitates Transcription Factor Wozniak, R. W., and Aitchison, J. Binding to Enhancers in Mature D. (2011) Role of the nuclear Cells. J Biol Chem 291, 18058- envelope in genome organization 18071 and gene expression. Wiley 60. Turriziani, B., Garcia-Munoz, A., Interdiscip Rev Syst Biol Med 3, Pilkington, R., Raso, C., Kolch, 147-166 W., and von Kriegsheim, A. 57. Canel, M., Byron, A., Sims, A. H., (2014) On-beads digestion in Cartier, J., Patel, H., Frame, M. conjunction with data-dependent C., Brunton, V. G., Serrels, B., mass spectrometry: a shortcut to and Serrels, A. (2017) Nuclear quantitative and dynamic FAK and Runx1 Cooperate to interaction proteomics. Biology 3, Regulate IGFBP3, Cell-Cycle 320-332 Progression, and Tumor Growth. Cancer research 77, 5301-5312

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899328; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.