The innate immune response is a primary transcriptional defence network activated by interferons (IFNs) α/ β in response to viral infection. A cell must have the capability to detect the virus, activate signalling cascades, and engage transcriptional anti-viral networks. IFNs trigger the Signal Transducer and Activator of Transcription (STAT) family, which in turn induce anti-viral gene expression. Recruitment of STATs to IFN stimulated gene (ISG) promoters and the ensuing gene induction requires Brahma Related Gene 1 (BRG1), the catalytic component of the SWI/ SNF chromatin remodelling (or BAF) complex. Cell lines with high BRG1 expression are hyper-responsive to IFN induced transcription, conversely BRG1 low cells exhibit impaired induction. However, BRG1 high cells that are resistant to Encephalomyocarditis virus infection did not require signalling through the IFN receptor complex for anti-viral immunity. This suggested 2F-BRG1 cells must rely on BRG1 dependent non-ISGs or an as yet uncharacterized subset of basally expressed BRG1-dependent ISGs that do not require IFN enhanced expression for anti-viral activity. Utilizing genome wide microarrays we identified five genes with potent anti-viral activity. These genes may restrict viral infection through alterations in integrin signalling, endosomal trafficking, and activation of host transcriptional responses. We also investigated the role of Special AT-Rich Binding Protein (SATB1) in regulation of IFN responsive genes. The loss of this chromatin binding protein is associated with transcriptional changes in the MHC locus that mimic IFNγ induced expression. Through microarray analysis we discovered a remarkable 47% of IFNα regulated genes were co-regulated by SATB1; 42% of IFNα induced genes were induced by SATB1 knock down, while 63% of IFNα repressed genes were SATB1 dependent. Functionally, knock down of SATB1 protected cells from EMCV induced cell death at low multiplicity of infection (MOI), and increased the cytoprotective effect of IFNα against EMCV at higher MOIs. Analysis of IFNα, SATB1 and BRG1 regulated genes revealed a subset of core genes regulated by all three factors that may be critical to robust anti-viral immunity. The potent immunosuppressive properties of SATB1 suggest this protein may be involved in complex immunopathologies. The immuno-modulatory properties of SATB1 and BRG1 established in this thesis provide substantive evidence for the development of pharmaceutical therapies targeting these proteins.

The innate immune response is a primary transcriptional defence network activated by interferons (IFNs) α/ β in response to viral infection. A cell must have the capability to detect the virus, activate signalling cascades, and engage transcriptional anti-viral networks. IFNs trigger the Signal Transducer and Activator of Transcription (STAT) family, which in turn induce anti-viral gene expression. Recruitment of STATs to IFN stimulated gene (ISG) promoters and the ensuing gene induction requires Brahma Related Gene 1 (BRG1), the catalytic component of the SWI/ SNF chromatin remodelling (or BAF) complex. Cell lines with high BRG1 expression are hyper-responsive to IFN induced transcription, conversely BRG1 low cells exhibit impaired induction. However, BRG1 high cells that are resistant to Encephalomyocarditis virus infection did not require signalling through the IFN receptor complex for anti-viral immunity. This suggested 2F-BRG1 cells must rely on BRG1 dependent non-ISGs or an as yet uncharacterized subset of basally expressed BRG1-dependent ISGs that do not require IFN enhanced expression for anti-viral activity. Utilizing genome wide microarrays we identified five genes with potent anti-viral activity. These genes may restrict viral infection through alterations in integrin signalling, endosomal trafficking, and activation of host transcriptional responses. We also investigated the role of Special AT-Rich Binding Protein (SATB1) in regulation of IFN responsive genes. The loss of this chromatin binding protein is associated with transcriptional changes in the MHC locus that mimic IFNγ induced expression. Through microarray analysis we discovered a remarkable 47% of IFNα regulated genes were co-regulated by SATB1; 42% of IFNα induced genes were induced by SATB1 knock down, while 63% of IFNα repressed genes were SATB1 dependent. Functionally, knock down of SATB1 protected cells from EMCV induced cell death at low multiplicity of infection (MOI), and increased the cytoprotective effect of IFNα against EMCV at higher MOIs. Analysis of IFNα, SATB1 and BRG1 regulated genes revealed a subset of core genes regulated by all three factors that may be critical to robust anti-viral immunity. The potent immunosuppressive properties of SATB1 suggest this protein may be involved in complex immunopathologies. The immuno-modulatory properties of SATB1 and BRG1 established in this thesis provide substantive evidence for the development of pharmaceutical therapies targeting these proteins.

Yes / Nottingham Trent University, United Kingdom, UoA03 QR and Capital Funds (MIA), as well as by the grant from Amway, USA to VAB and NVB.

No description available.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

Squamous cell carcinoma of the head and neck region (HNSCC) is an aggressive malignancy with generally poor prognosis and high mortality. The Special AT-rich binding protein 1 (SATB1) is a genome organizer protein that participates in regulating gene expression by acting as a trans-acting element as well as by recruiting chromatin remodeling complexes and enzymes. SATB1 is often overexpressed in cancer, and its possible role in tumour progression has been explored in several types of cancers and also suggested in HNSCC. However, its influence on molecular and cellular processes in HNSCC has not been examined, and, using primary cell lines, provided the basis of this thesis. This is a comprehensive study of molecular and cellular processes being affected upon siRNA-mediated SATB1 knockdown in vitro and in vivo. 15 HNSCC primary cell lines were obtained from the University of Turku and screened for SATB1 mRNA levels. The comparison of SATB1 mRNA levels with location, lymph node metastasis, disease staging (TNM) or SATB2 mRNA levels revealed no association. Hence, for deeper analysis 7 primary cell lines were selected based on growth inhibitory effects upon transient SATB1 knockdown, rather than their initial SATB1 mRNA levels. Growth inhibition upon SATB1 depletion was shown in monolayer (viable cell quantitation and colony forming ability) as well as non-adherent (spheroid assay) culture conditions. In some cell lines, cell death induced by apoptosis or retardation of cell cycle progression was observed as well. Parallel to this, using the FLAVINO assay, colony forming abilities of tumour cells from patient biopsies obtained from the University Hospital of Leipzig (Department of Otorhinolaryngology, Head and Neck Surgery) were tested post SATB1 knockdown. For molecular analysis, effects of SATB1 knockdown on transcription rates of selected oncogenes were analyzed. Among EMT markers, N-cadherin and beta catenin levels were found reduced upon SATB1 knockdown. The transcription of HER3 and its ligands Heregulin α & β was attenuated in all the seven primary cell lines, irrespectively of the growth inhibitory effects of SATB1 knockdown. These results demonstrated the role of SATB1 in the process of EMT and in autocrine signalling. Effects of HER3 inhibition on transcription rates of SATB1 were tested as well. HER3 inhibition was achieved by Patritumab, a novel monoclonal antibody against HER3. While SATB1 transcription rates remained unchanged upon HER3 inhibition, growth inhibition assays (2D and 3D) revealed that the combined use of HER1 and HER3 inhibitory antibodies provides better tumour cell inhibition over the single treatment. Finally, antitumor effects of SATB1 knockdown were monitored in vivo in two xenograft models (UT-SCC-14 and UT-SCC-42B). Treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles revealed reduced tumour growth, based on the knockdown of SATB1 as demonstrated on the protein level. Taken together, in this work SATB1 knockdown is demonstrated to mediate growth inhibition, induction of apoptosis, cell cycle retardation, negative impact on EMT and autocrine signaling and in vivo anti-tumour effects, thus highlighting the relevance of SATB1 in HNSCC.:156 pages

Kolorektal cancer (CRC) är en av de vanligaste cancersjukdomarna i världen med cirka 1 miljon nya detekterade fall per år. Special AT-rich sequence-binding protein1 (SATB1) är ett celltyp-specifikt kärnmatrix-associerat DNA-bindande protein, vilket utgörs av AT-rika DNA sekvenser. Det har tidigare demonstrerats att en annan medlem i SATB-familjen, SATB2, uttrycks på ett vävnadsspecifikt sätt i normal mukosa i nedre mag-tarmkanalen och i CRC. β-catenin är en intracellulär mediator i Wnt/β-catenin signaleringsvägen, som spelar en viktig roll i kolorektal carcinogenes. Uttryck av SATB1, SATB2 och β-catenin har studerats i tissue microarrays med tumörprover från 270 CRC patienter. Deras inbördes korrelation samt koppling till recidivfri överlevnad har studerats med hjälp av Spearman´s korrelationstest respektive Kaplan-Meier analys och log-rank test. Resultatet från immunhistokemiska färgningar visar att det finns en korrelation mellan de analyserade markörerna. Därutöver fann vi att SATB1 uttryck är kopplat till kortare recidivfri överlevnad i tumörer med lågt SATB2 uttryck. / Colorectal cancer (CRC) is one of the most common cancers in the world with about 1 million new cases annually. Special AT-rich sequence binding protein 1 (SATB1), is a cell type specific nuclear matrix associated DNA binding protein, which consists of AT-rich DNA sequences. It has previously been demonstrated that another member in SATB-family, SATB2, is expressed in a tissue-specific manner in normal mucosa in the lower gastrointestinal tract and in CRC. β-catenin is an intracellular mediator of the Wnt/ β-catenin signaling pathway and plays an important role in colorectal carcinogenesis. Expression of SATB1, SATB2 and β-catenin was analyzed in tissue microarrays with tumors from 270 CRC patients. Spearman´s correlation test was used to assess the correlations and the impact of SATB1 and SATB2 on recurrence free survival was assessed by Kaplan-Meier analysis and log-rank test. The result of immunohistochemical staining shows that there is a correlation between the analyzed markers and that SATB1 expression is a poor prognostic factor in tumors expressing low levels of SATB2.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

MET-amplified gastric cancer cells are extremely sensitive to MET inhibition in vitro, whereas clinical efficacy of MET inhibitors is disappointing. The compensatory activation of other oncogenic growth factor receptors may serve as an underlying mechanism of resistance. In this study, we analyzed the role of HER receptors, in particular HER3 and its ligand heregulin, in this respect. This also included the chromatin-organizer protein SATB1, as an established regulator of HER expression in other tumor entities. In a panel of MET-amplified gastric carcinoma cell lines, cell growth under anchorage-dependent and independent conditions was studied upon inhibitor treatment or siRNA-mediated knockdown. Expression analyses were performed using RT-qPCR, FACS, and immunoblots. Signal transduction was monitored via antibody arrays and immunoblots. As expected, MET inhibition led to a growth arrest and inhibition of MAPK signaling. Strikingly, however, this was accompanied by a rapid and profound upregulation of the oncogenic receptor HER3. This finding was determined as functionally relevant, since HER3 activation by HRG led to partial MET inhibitor resistance, and MAPK/Akt signaling was even found enhanced upon HRG+MET inhibitor treatment compared to HRG alone. SATB1 was identified as mediator of HER3 upregulation. Concomitantly, SATB1 knockdown prevented upregulation of HER3, thus abrogating the HRG-promoted rescue from MET inhibition. Taken together, our results introduce the combined HER3/MET inhibition as strategy to overcome resistance towards MET inhibitors.

Overexpression of members of the HER/erbB transmembrane tyrosine kinase family like HER2/erbB2/neu is associated with various cancers. Some heterodimers, especially HER2/HER3 heterodimers, are particularly potent inducers of oncogenic signaling. Still, from a clinical viewpoint their inhibition has yielded only moderate success so far, despite promising data from cell cultures. This suggests acquired resistance upon inhibitor therapy as one putative issue, requiring further studies in cell culture also aiming at rational combination therapies. In this paper, we demonstrate in ovarian carcinoma cells that the RNAi-mediated single knockdown of HER2 or HER3 leads to the rapid counter-upregulation of the respective other HER family member, thus providing a rational basis for combinatorial inhibition. Concomitantly, combined knockdown of HER2/HER3 exerts stronger anti-tumor effects as compared to single inhibition. In a tumor cell line xenograft mouse model, therapeutic intervention with nanoscale complexes based on polyethylenimine (PEI) for siRNA delivery, again reveals HER3 upregulation upon HER2 single knockdown and a therapeutic benefit from combination therapy. On the mechanistic side, we demonstrate that HER2 knockdown or inhibition reduces miR-143 levels with subsequent de-repression of HER3 expression, and validates HER3 as a direct target of miR-143. HER3 knockdown or inhibition, in turn, increases HER2 expression through the upregulation of the transcriptional regulator SATB1. These counter-upregulation processes of HER family members are thus based on distinct molecular mechanisms and may provide the basis for the rational combination of inhibitors.