Y-box binding protein 1 (YB-1) relevance in estrogen receptor-positive (ER+) breast cancer

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016

The role of Y-box binding protein 1 in prostate cancer

This thesis examined the possible role of Y-box binding protein 1 (YBX1) in prostate cancer aggression and spread. Novel roles were uncovered for YBX1 in the regulation of several genes previously implicated in prostate cancer, as well as showing an effect for YBX1 in increasing tumour cell invasion and movement and reciprocal regulation of androgen-regulated gene networks. In addition, it was found that Y-box 1 regulated several other well-known cancer genes implicated in breast and other cancers. The work performed in this thesis has strengthened the foundations for pursuing YBX1 as a possible central target molecule in prostate cancer therapeutics.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3′→5′ exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation

We have recently demonstrated that thrombin induces expression of the platelet-derived growth factor B-chain gene in endothelial cells (EC) through activation of the Y-box binding protein DNA-binding protein B (dbpB). We now present evidence that dbpB is activated by a novel mechanism: proteolytic cleavage leading to release from mRNA, nuclear translocation, and induction of thrombin-responsive genes. Cytosolic, full-length dbpB (50 kDa) was rapidly cleaved to a 30-kDa species upon thrombin stimulation of EC. This truncated, “active” dbpB exhibited nuclear localization and binding affinity for the thrombin response element sequence, which is distinct from the Y-box sequence. Oligo(dT) affinity chromatography revealed that cytosolic dbpB from control EC, but not active dbpB from thrombin-treated EC, was bound to mRNA. Latent dbpB immunoprecipitated from cytosolic extracts of control EC was activated by ribonuclease treatment. Furthermore, when EC cytosolic extracts were subjected to Nycodenz gradient centrifugation, latent dbpB fractionated with mRNA, whereas active dbpB fractionated with free proteins. The cytosolic retention domain of dbpB, which we localized to the region 247–267, was proteolytically cleaved during its activation. In contrast to full-length dbpB, truncated dbpB stimulated platelet-derived growth factor B-chain and tissue factor promoter activity by over 5-fold when transiently cotransfected with reporter constructs. These results suggest a novel mode of transcription factor activation in which an agonist causes release from mRNA of a latent transcription factor leading to its transport to the nucleus and its regulation of target gene expression.

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well-known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a vascular endothelial growth factor receptor (VEGFR) and PI3K/Akt dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Over-expression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 Protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3 and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^

Multiple Genes Encoding the Conserved CCAAT-Box Transcription Factor Complex Are Expressed in Arabidopsis

The CCAAT motif is found in the promoters of many eukaryotic genes. In yeast a single complex of three proteins, termed HAP2, HAP3, and HAP5, binds to this sequence, and in mammals the three components of the equivalent complex (called variously NF-Y, CBF, or CP1) are also represented by single genes. Here we report the presence of multiple genes for each of the components of the CCAAT-binding complex, HAP2,3,5, from Arabidopsis. Three independent Arabidopsis HAP subunit 2 (AtHAP2) cDNAs were cloned by functional complementation of a yeast hap2 mutant, and two independent forms each of AtHAP3 and AtHAP5 cDNAs were detected in the expressed sequence tag database. Additional homologs (two of AtHAP3 and one of AtHAP5) have been identified from available Arabidopsis genomic sequences. Northern-blot analysis indicated ubiquitous expression for each AtHAP2 and AtHAP5 cDNA in a range of tissues, whereas expression of each AtHAP3 cDNA was under developmental and/or environmental regulation. The unexpected presence of multiple forms of each HAP homolog in Arabidopsis, compared with the single genes in yeast and vertebrates, suggests that the HAP2,3,5 complex may play diverse roles in gene transcription in higher plants.

Core promoter-specific function of a mutant transcription factor TFIID defective in TATA-box binding.

In conjunction with other general initiation factors, the TATA box-binding protein (TBP) can direct basal transcription by RNA polymerase II from TATA-containing promoters, but its stable interaction with TBP-associated factors (TAFs) in the TFIID complex is required both for activator-dependent transcription and for basal transcription directed by an initiator element. We have generated a TATA-binding-defective TFIID complex containing an amino acid substitution in the DNA-binding surface of its TBP subunit. This mutated TFIID is defective in both basal and activated transcription from core promoters containing only a TATA box but supports transcription from initiator-containing promoters independently of the presence or absence of a TATA sequence. Our results show that a functional initiator element is needed to bypass the requirement for an active TATA DNA-binding surface in TFIID and imply that gene-specific transcription can be achieved by modulating distinct core promoter-specific TFIID functions--e.g., TBP-TATA versus TAF-initiator interactions.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

The molecular and genetic characterization of the MADS box transcription factor {\it Drosophila\/} MEF2

The myocyte enhancer factor (MEF)-2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates, but the precise position of these regulators within the genetic hierarchy leading to myogenesis is unclear. The MEF2 proteins bind to a conserved A/T-rich DNA sequence present in numerous muscle-specific genes, and they are expressed in the cells of the developing somites and in the embryonic heart at the onset of muscle formation in mammals. The MEF2 genes belong to the MADS box family of transcription factors, which control specific programs of gene expression in species ranging from yeast to humans. Each MEF2 family member contains two highly conserved protein motifs, the MADS domain and the MEF2-specific domain, which together provide the MEF2 factors with their unique DNA binding and dimerization properties. In an effort to further define the function of the MEF2 proteins, and to evaluate the degree of conservation shared among these factors and the phylogenetic pathways that they regulate, we sought to identify MEF2 family members in other species. In Drosophila, a homolog of the vertebrate MEF2 genes was identified and termed D-mef2. The D-MEF2 protein binds to the consensus MEF2 element and can activate transcription through tandem copies of that site. During Drosophila embryogenesis, D-MEF2 is specific to the mesoderm germ layer of the developing embryo and becomes expressed in all muscle cell types within the embryo. The role of D-mef2 in Drosophila embryogenesis was examined by generating a loss-of-function mutation in the D-mef2 gene. In embryos homozygous for this mutant allele, somatic, cardiac, and visceral muscles fail to differentiate, but precursors of these myogenic lineages are normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2 and suggest that this program has been conserved from Drosophila to mammals. ^

Mechanism of subunit interaction and DNA binding of the heterotrimeric CCAAT binding factor CBF

Many eukaryotic promoters contain a CCAAT element at a site close ($-$80 to $-$120) to the transcription initiation site. CBF (CCAAT Binding Factor), also called NF-Y and CP1, was initially identified as a transcription factor binding to such sites in the promoters of the Type I collagen, albumin and MHC class II genes. CBF is a heteromeric transcription factor and purification and cloning of two of the subunits, CBF-A and CBF-B revealed that it was evolutionarily conserved with striking sequence identities with the yeast polypeptides HAP3 and HAP2, which are components of a CCAAT binding factor in yeast. Recombinant CBF-A and CBF-B however failed to bind to DNA containing CCAAT sequences. Biochemical experiments led to the identification of a third subunit, CBF-C which co-purified with CBF-A and complemented the DNA binding of recombinant CBF-A and CBF-B. We have recently isolated CBF-C cDNAs and have shown that bacterially expressed purified CBF-C binds to CCAAT containing DNA in the presence of recombinant CBF-A and CBF-B. Our experiments also show that a single molecule each of all the three subunits are present in the protein-DNA complex. Interestingly, CBF-C is also evolutionarily conserved and the conserved domain between CBF-C and its yeast homolog HAP5 is sufficient for CBF-C activity. Using GST-pulldown experiments we have demonstrated the existence of protein-protein interaction between CBF-A and CBF-C in the absence of CBF-B and DNA. CBF-B on other hand, requires both CBF-A and CBF-C to form a ternary complex which then binds to DNA. Mutational studies of CBF-A have revealed different domains of the protein which are involved in CBF-C interaction and CBF-B interaction. In addition, CBF-A harbors a domain which is involved in DNA recognition along with CBF-B. Dominant negative analogs of CBF-A have also substantiated our initial observation of assembly of CBF subunits. Our studies define a novel DNA binding structure of heterotrimeric CBF, where the three subunits of CBF follow a particular pathway of assembly of subunits that leads to CBF binding to DNA and activating transcription. ^

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.