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Genes, pathways & transcription factors involved in probiotic mediated resolution of gut inflammation in IL10-KO mice an animal model of Inflammatory Bowel Disease. An integrated Gene, Protein and Bioinformatics Approach The IL10-KO mouse is a model of human inflammatory bowel disease (IBD), used to study host microbial interactions and potential therapeutics. Affymetrix microarray and proteomics analysis on colon of WT and IL10-KO mice and cecum of IL10-KO and WT mice orally administered with and without probiotic VSL#3 was performed and identified signalling pathways and transcription factors relevant to gut inflammation and anti-inflammatory probiotics. Results were validated by Real-time PCR, immunocytochemistry, proteomics, histopathology and via pathway signature analysis of publicly available microarray data. Changes in metabolically active bacteria in response to VSL#3 were assessed with DGGE. Inflammation in IL10-KO mice was characterised by up-regulation of immune/inflammatory and down-regulation of lipid/xenobiotic metabolism and PPAR signalling. VSL#3 resolved inflammation in the cecum inducing down-regulation of genes in immune/inflammatory pathways, decrease in the number of CCL5 positive T cells and up-regulation of galectin2, known to trigger apoptosis of T cells. VSL#3 induced up-regulation of PPARα/PPAR signalling and lipid/xenobiotic metabolism, antagonistic to NFB signalling and reduced metabolically active bifidobacteria. Analysis of publicly available data showed results were relevant to human IBD, indicated that antigen processing/presentation is up-regulated early on during development of colitis in IL10-KO mice, identified the potential of PPARα/PPAR signalling to induce down-regulation of CCL5, CD3 & antigen processing/presentation, and the potential of the xenobiotic metabolism to induce down-regulation of cytokine-cytokine interaction & mitosis. As VSL#3 treatment of IL10-KO mice induced up-regulation of PPARα/PPAR signalling and xenobiotic metabolism these results provide a possible mechanistic explanation for the VSL#3 induced down-regulation of CCL5, CD3, antigen processing/presentation, cytokine-cytokine interaction and mitosis in the cecum of VSL#3 treated IL10-KO mice.

The p53 family of transcription factors, which comprises the products of the TP53, TP63 and TP73 genes, is at the hub of different signalling pathways that determine the fate of a cell. In vitro, the p53 family posses a tumour suppressive function. However, in vivo, although p53-deficient mice develop spontaneous tumours, p73 and p63 KO animals show defects in neuronal and epidermal development, respectively. The ASPP family of proteins also consists of three members: ASPP 1, ASPP2 and iASPP. They are evolutionarily conserved binding partners and specific regulators of p53-, p63- and p73- mediated apoptosis in vitro. In contrast to ASPPl and ASPP2, the biological significance of iASPP in vivo and the possible interaction with p53 family members remains unclear, and it is the subject of this study. To investigate the role of iASPP in vivo, a transgenic mouse model was generated in which iASPP expression is controlled by the Cre/loxP recombination system. Deletion of iASPP resulted in the development of a cardiocutaneous disorder which displayed features of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), defects in hair follicle position and impaired epithelial stratification. iASPP loss resulted in a sudden, premature and arrhythmic mode of death of all iASPP mutant mice. iASPP deficiency induced p53-dependent apoptosis in embryonic hearts and dilation of the right ventricle, however, the inactivation of p53 alleles only rescued the fibro-fatty deposits present in iASPP KO hearts. Mechanistically, iASPP locates at the polar ends of cardiomyocytes where it matches the location of other proteins known to be involved in the etiology of ARVC and in maintaining the integrity of intercalated discs. Loss of iASPP also resulted in increased differentiation of primary keratinocytes both in vitro and in vivo. Consistent with this, iASPP bound p63 and inhibited the transcriptional activity of both T Ap63a and T Ap63α and ΔNp63 in vitro, and regulated the expression level ofp63-regulated genes such as envoplakin. These results demonstrate that iASPP prevents cardiocutaneous disorder through its ability to inhibit p53-induced apoptosis and to influence the integrity of intercalated disc. Moreover, iASPP is also an important regulator of p63 and is involved in controlling epithelial stratification in vivo.

Regulation of gene expression in a spatiotemporal manner specifies cellular identity. Transcription factors (TFs) bind to DNA regulatory elements to remodel chromosome structure, to recruit transcription machinery to initiate gene transcription or to prevent the assembly of such machinery to repress gene transcription, thus they lie at the heart of gene regulation. Given important roles of TFs in gene regulation, numerous attentions have been attracted for engineered transcription factors (eTFs). The recent advance of generating customized DNA-sequence specific binding domains, including transcription activator-like effectors (TALEs) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene Cas9, has greatly accelerated the study and application of eTFs. The eTFs with these new binding domains offer a powerful and precise approach for modulating gene expression. / Oct4 is an important TF and it plays essential roles in the formation of inner cell mass during embryogenesis, and the maintenance of embryonic stem cells in culture as well as the reinstatement of cellular pluripotency from somatic cells. / In this study, we systematically investigated the potential of TALE-TFs and CRISPR/Cas9-TFs in activating Oct4. We designed a number of TALEs and small guide RNAs (sgRNAs) targeting various regions in the mouse and human Oct4 promoters. Using luciferase assays, we found that the most efficient TALE-VP64s bound on the region −120 to −80 bp upstream of transcription start site (TSS), while highly effective sgRNAs targeted −147 to −89 bp upstream of TSS to induce high activity of luciferase reporters. This positional effect can serve as a simple guideline for designing eTFs for activating transcription from a reporter system. Next, we examined the potential of TALE-VP64 and sgRNAs to activate endogenous Oct4 transcription. We found that the positional effect was less obvious as individual eTFs exhibited marginal activity to up-regulate endogenous gene expression. Interestingly, we found that when multiple eTFs were applied simultaneously, Oct4 could be induced significantly and synergistically. This phenomenon was well supported by activation of human SOX2, KLF4, cMYC, CDH1 and NANOG by TALE-VP64s. / Using optimized combinations of TALE-VP64s, we successfully enhanced endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64- and sgRNA/dCas9-VP64-induced transcription of endogenous OCT4. Taken together, this study demonstrated that engineered TALE-TFs and dCas9-TFs are useful tools for modulating gene expression in mammalian cells. / 基因表達調控是決定細胞命運的關鍵。轉錄因子可以結合到DNA調控序列上，以重塑染色體的結構；而且可以募集轉錄機器，以起始轉錄, 或者幹擾轉錄機器的組裝，從而抑制基因轉錄；因此，在基因表達調控過程中轉錄因子處於核心地位。由于轉錄因子在基因調控方面的重要作用，研究者們越來越多的關注人工轉錄因子的研究。DNA 序列特異性結合域的發現與發展很大程度上促進了人工轉錄因子的研究與應用。最近從TALE和CRISPR/Cas9衍生而來的人工轉錄因子給我們提供了一個強大而且精確的調控基因表達的方法。Oct4是一個重要的轉錄因子，對胚胎發育過程中內細胞團的形成，和體外培養的胚胎幹細胞的維持，以及細胞多能性的重塑等多方面都至關重要。 / 在本研究中，我們系統性地探討了TALE和CRISPR/Cas9衍生而來的人工轉錄因子在激活Oct4基因方面的潛能。我們針對小鼠和人的Oct4的啓動子設計了一序列的TALEs和sgRNAs。通過熒光素酶實驗，我們發現結合到轉錄起始位點上遊120‐80bp位置的TALE‐VP64s，或者結合到147‐89bp位置的sgRNAs可以最有效地誘導熒光素酶報告基因的表達。在激活報告基因方面，這種位置效應可以作爲一條設計人工轉錄因子的簡單原則。然後，我們進一步檢測了這些人工轉錄因子在激活內源性Oct4轉錄方面的效果。結果顯示上述觀察到的位置效應並不明顯，因爲每一單個的人工轉錄因子都幾乎不能上調內源性基因的表達。但是，當同時導入多個人工轉錄因子時，我們可以顯著地激活Oct4的表達，而且可以觀察到明顯的疊加效應。利用人工轉錄因子激活SOX2, KLF4, cMYC, CDH1和NANOG,我們進一步證明了這種疊加效應。 / 通過篩查不同的人工轉錄因子組合，我們在小鼠NIH3T3細胞系把Oct4基因的表達提供到了原來水平的30多倍，而在人的HEK293T中，提高了20多倍。更重要的是，我們可以檢測到蛋白質表達水平的提高。通過檢測不同的表觀調控因子，我們發現組蛋白乙酰化轉移酶p300可以進一步提升這些人工轉錄因子誘導的Oct4基因表達。因此，本研究表明這些人工轉錄因子是調節哺乳動物細胞內基因表達的有效工具。 / Hu, Jiabiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.y066 / Includes bibliographical references (leaves 132-157). / Abstracts also in Chinese. / Title from PDF title page (viewed on 13, December, 2016). / Hu, Jiabiao. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

The nervous system is required for human cognition, motor function, and sensory interaction. A complex network of neuronal connections, or synapses, carries out these behaviors, and defects in neural connectivity can result in developmental and degenerative diseases. In vertebrate nervous systems, synapses most commonly occur at axon terminals. Upon reaching their synaptic targets, growth cones lose their motility and become boutons specialized for neurotransmitter release. I am studying this process in R7 photoreceptors in the

Transcription factors (TFs) bind to specific DNA sequences near target genes to precisely coordinate their regulation. Despite the central role of transcription factors in development and homeostasis, the mechanisms by which TFs have evolved to bind and regulate distinct DNA sequences are poorly understood. This dissertation details the highly collaborative work to determine the genetic, biochemical and biophysical mechanisms by which distinct DNA-binding specificities evolved in the steroid receptor (SR) family of transcription factors. Using ancestral protein reconstruction, we resurrected and functionally characterized the historical transition in DNA-binding specificity between ancient SR proteins. We found that DNA-binding specificity evolved by changes in the energetic components of binding; interactions at the protein-DNA interface were weakened while inter-protein cooperativity was greatly improved. We identified a group of fourteen historical substitutions that were sufficient to recapitulate the derived protein's binding function. Three of these substitutions, which we defined as function-switching, were sufficient to change DNA specificity; however, their introduction greatly decreased binding affinity and was deleterious for protein function. A group of eleven permissive substitutions, which had no effect on DNA specificity, allowed for the protein to tolerate the deleterious effects of the function-switching substitutions. They non-specifically increased binding affinity by improving interactions at the protein-DNA interface and increasing inter-protein cooperativity. We then dissected the functional role of individual substitutions in both the function-switching and permissive groups. We first determined the binding affinity of all possible combinations of function-switching substitutions for a library of DNA sequences. This allowed for us to functionally characterize the sequence space that separated the ancestral and derived DNA-binding specificities as well as identify the genetic determinants for DNA specificity. Lastly, we dissected the effects of the permissive substitutions on the energetics of DNA binding to determine the mechanisms by which they exerted their permissive effect. Together, this work provides insight into the molecular determinants of DNA specificity and identifies the molecular mechanisms by which these interactions changed during the evolution of novel specificity in an important transcription factor family. This dissertation includes previously published and unpublished co-authored material. / 2016-01-14

Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis fails to consistently induce sufficient reperfusion to facilitate repair, targeted manipulation of angiogenesis is clinically desirable. The Wilms' tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that WT1 in the endothelium influences the angiogenic function of endothelial cells. WT1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from WT1-GFP reporter mice. Inducible deletion of WT1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p < 0.05 relative to controls). In vivo, WT1 expression was evident in vascular endothelial and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia, often in cells expressing epithelial and mesenchymal markers simultaneously. However, VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density or expression of angiogenic factors (qRT-PCR) in sponges inserted subcutaneously in mice (20 days). To further understand the role of WT1 in angiogenesis, transcriptomic RNA expression analysis was performed in WT1+ and WT1- cells isolated (FACs) from sponges after implantation in WT1-GFP mice. WT1+ cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including angiogenesis (p=3.11x10-8), wound healing (p=3.45x10-7) and epithelial-to-mesenchymal transition (EMT) (p=5.86x10-4). These results shed new light on the role of WT1 in ischaemic angiogenesis. In concurrence with previously published work, we show that deletion of endothelial WT1 can delay angiogenesis however, WT1 is not just instrumental in endothelial cells in this context. WT1 has a broader role in tissue repair in ischaemia, in part through regulation of cell transition (EMT). This work has improved our understanding of the regulatory role of WT1 in angiogenesis and repair, while revealing a number of novel insights into the function of WT1. This highlights WT1 as a potentially beneficial therapeutic target to facilitate regeneration in cardiovascular disease.

by Sham Lok To. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 143-171). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / List of figures --- p.ix / List of tables --- p.xi / Chapter Chapter One --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- About L. edodes --- p.3 / Chapter 1.1.2 --- Nutritional and medicinal values of L. edodes --- p.4 / Chapter 1.1.3 --- Life cycle of L. edodes --- p.6 / Chapter 1.1.4 --- Environmental factors affecting fruiting body formation in L. edodes --- p.6 / Chapter 1.2 --- Molecular mechanisms of fruiting body development in L. edodes --- p.8 / Chapter 1.2.1 --- Expression profiling and identification of differentially expressed genes during fruiting --- p.8 / Chapter 1.2.2 --- Changing in membrane structure --- p.11 / Chapter 1.2.3 --- The signal transduction cascade --- p.12 / Chapter 1.3 --- Transformation in L. edodes and in other fungi --- p.14 / Chapter 1.3.1 --- Transformation of L. edodes --- p.14 / Chapter 1.3.2 --- Transformation in other fungi --- p.17 / Chapter 1.4 --- Bioinformatics tools for comparative promoter analysis --- p.22 / Chapter 1.5 --- Objectives and significance --- p.26 / Chapter Chapter Two --- Promoter analysis of differentially expressed genes (DEGs) in the fruiting body development in L. edodes --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and methods --- p.29 / Chapter 2.2.1 --- Strains and cultivation conditions --- p.29 / Chapter 2.2.2 --- Genome walking of the 5' flanking region of the DEGs --- p.29 / Chapter 2.2.3 --- Annealing Control Primed (ACP) PCR --- p.31 / Chapter 2.2.4 --- Construction of genomic DNA library --- p.36 / Chapter 2.2.5 --- Nested PCR to amplify the target sequences --- p.37 / Chapter 2.2.6 --- Cloning and sequencing of the 5' flanking region --- p.38 / Chapter 2.2.7 --- Determination of transcription start site by the Neural Network algorithm --- p.39 / Chapter 2.2.8 --- Identification of putative transcription factor binding sites --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Construction of adaptor linked template for genome walking --- p.41 / Chapter 2.3.2 --- Sequence analysis and quality control --- p.41 / Chapter 2.3.3 --- Comparison of various methods in genome walking --- p.42 / Chapter 2.3.4 --- Promoter analysis --- p.42 / Chapter 2.4 --- Discussion --- p.58 / Chapter Chapter Three --- In-silico analysis of transcription factor binding sites and identification transcription factors expressed in L. edodes --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Material and methods --- p.67 / Chapter 3.2.1 --- Sequence manipulation and extraction of homologous ESTs from C. cinereus --- p.67 / Chapter 3.2.2 --- Extraction of 5' flanking region of the corresponding ESTs and promoter prediction --- p.67 / Chapter 3.2.3 --- Positional cloning of mating type factor A --- p.68 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- Sequence extraction and manipulation --- p.70 / Chapter 3.3.2 --- In-silico analysis of transcription factor binding sites in C. cinereus . --- p.70 / Chapter 3.3.3 --- Comparison of putative TFBS between L. edodes and C. cinereus --- p.71 / Chapter 3.3.4 --- Identification of transcription factors in L. edodes by positional cloning --- p.71 / Chapter 3.4 --- Discussion --- p.85 / Chapter Chapter Four --- Identification，expression profiling and promoter analysis of hydrophobin genes --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Material and methods --- p.92 / Chapter 4.2.1 --- Clustering and grouping of the hydrophobin ESTs --- p.92 / Chapter 4.2.2 --- Identification of the consensus sequences of the hydrophobin groups --- p.93 / Chapter 4.2.3 --- RNA Sources and Preparation --- p.93 / Chapter 4.2.4 --- Expression profiling of hydrophobin genes by RT-PCR --- p.95 / Chapter 4.2.5 --- Promoter cloning and analysis of hydrophobin genes --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Isolation and characterization of four newly found hydrophobin genes --- p.97 / Chapter 4.3.2 --- Expression levels of hydrophobins --- p.100 / Chapter 4.3.3 --- Promoter sequencing of the hydrophobins --- p.103 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter Five --- Transformation of L. edodes --- p.110 / Chapter 5.1 --- Introduction --- p.110 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Vectors and primers design --- p.112 / Chapter 5.2.2 --- Maxi-preparation of plasmids --- p.112 / Chapter 5.2.3 --- Cultural condition and optimization of protoplasts release --- p.114 / Chapter 5.2.4 --- PEG mediated transformation --- p.115 / Chapter 5.2.5 --- Electroporation mediated transformation --- p.116 / Chapter 5.2.6 --- PCR screening of regenerated transformant --- p.116 / Chapter 5.2.7 --- Particle bombardment --- p.117 / Chapter 5.3 --- Results --- p.121 / Chapter 5.4 --- Discussion --- p.128 / Chapter Chapter Six --- General discussions --- p.132 / References --- p.143

"Histone deacetylases (HDACs) have become one of the leading areas of research for cancer, neurodegenerative diseases, diabetes, obesity, and inflammation. Although HDACs are currently expressible in mammalian cultures and yeast, it is important to explore other cost effective options. Here it is shown that class I HDACs are expressible in insect cells. As well as expressing full length domains for class I HDACs, predicted active domains have also been expressed. This information can be utilized in many areas for future research including identifying unique sites to allow development of specific inhibitors for each HDAC, and developing a better understanding of the specific role of each HDAC. "