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Ulcerative colitis (UC) is a common disease with great variability in severity, with a high recurrence rate and heavy disease burden. In recent years, the different biological functions of competing endogenous RNA (ceRNA) networks of long noncoding RNAs (lncRNAs) and microRNAs (miRs) have aroused wide concerns, the ceRNA network of ulcerative colitis (UC) may have potential research value, and these expressed noncoding RNAs may be involved in the molecular basis of inflammation recurrence and progression. This study analyzed 490 colon samples associated with UC from 4 gene expression microarrays from the GEO database and identified gene modules by weighted correlation network analysis (WGCNA). CIBERSORT detected tissue-infiltrating leukocyte profiling by deconvolution of microarray data. LncBase and multiMIR were used to identify lncRNA-miRNA-mRNA interaction. We constructed a ceRNA network which includes 4 lncRNAs (SH3BP5-AS1, MIR4435-2HG, ENTPD1-AS1, and AC007750.1), 5 miRNAs (miR-141-3p, miR-191-5p, miR-192-5p, miR-194-5p, and miR196-5p), and 52 mRNAs. Those genes are involved in interleukin family signals, neutrophil degranulation, adaptive immunity, and cell adhesion pathways. lncRNA MIR4435-2HG is a variable in the decision tree for moderate-to-severe UC diagnostic prediction. Our work identifies potential regulated inflammation-related lncRNA-miRNA-mRNA regulatory axes. The regulatory axes are dysregulated during the deterioration of UC, suggesting that it is a risk factor for UC progression.

The tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression., Liou et al. report a 4.6 Å resolution structure of the human p53/ RNA polymerase II assembly, using single particle cryoelectron microscopy. This study suggests that p53’s functional domains regulate the DNA binding activity of RNA polymerase II, providing insights into p53-regulated gene expression.

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs before differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG, and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG, and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation.

To prevent tumorigenesis, p53 stimulates transcription by facilitating the recruitment of the transcription machinery on target gene promoters. Cryo-Electron Microscopy studies on p53-bound RNA Polymerase II (Pol II) reveal that p53 structurally regulates Pol II to affect its DNA binding and elongation, providing new insights into p53-mediated transcriptional regulation.

Perenniporiopsis gen. nov. is described to accommodate Perenniporiopsis minutissima spec. et comb. nov. Phylogenetic analyses based on two data sets, [ITS+n28S nrRNA] and [ITS, n28S, mtSSU, tef1], and using Maximum Parsimony, Maximum Likelihood and Bayesian inferences, showed that specimens of Perenniporia minutissima form a monophyletic well-supported clade within the core polyporoid clade, The new genus grouped with the Abundisporus, Perenniporiella and Perenniporia s.s. lineages, and is phylogenetically distinct from the Perenniporia s.s. lineage (100% BS, 99% MP, 1.00 BPP). Morphologically, the consistency of the basidiocarps, waxy when fresh, drying rigidly osseous, as well as the large basidiospores characterize this species and distinguish it from Perenniporia s.s. The species seems to be endemic to temperate east Asia, and is known from temperate Japan and China.

The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct yet poorly characterized interaction between p53 and RNA polymerase II (Pol II). Therefore, we dissected the human p53/Pol II interaction via single-particle cryo-electron microscopy, structural docking, and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA-binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA-binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by the position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA-binding functions of Pol II via the clamp domain, thereby providing insights into p53-regulated Pol II transcription.

The core promoter recognition TFIID complex acts as a central regulator for eukaryotic gene expression. To direct transcription initiation, TFIID binds the core promoter DNA and aids recruitment of the transcription machinery (e.g., RNA polymerase II) to the transcription start site. Many transcription factors target TFIID to control vital cellular processes. Current studies on finding TFIID interactors have predominantly focused on transcription factors. Yet, a comprehensive interactome of mammalian TFIID has not been established. Therefore, this study sought to reveal potential TFIID-nucleated networks by identifying likely co-regulatory factors that bind TFIID. By using intact native human TFIID complexes, we have exploited three independent approaches including a high-throughput Next Generation DNA sequencing coupled with proteomic analysis. Among these methods, we found some overlapping and new candidates in which we further assessed three putative interactors (i.e., Sox2, H2A and EMSY) by co-immunoprecipitation assays. Notably, in addition to known TFIID interactors, we identified a number of novel factors that participate either in co-regulatory pathways or non-transcription related functions of TFIID. Overal, these results indicate that, in addition to transcription initiation, mammalian TFIID may be involved in broader regulatory pathways than previous studies suggested.

Transcriptional bursting involves genes rapidly switching between active and inactive states. Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA Pol II during transcriptional bursting. It is unknown how acetylated chromatin is dynamically targeted and regulated by chromatin remodelers such as PBAF. Thus, we sought to understand how PBAF targets acetylated chromatin using live-cell single molecule fluorescence microscopy. Our work reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF’s bromodomains impairs targeting, stable engagement and persistent binding on chromatin in hubs. Interestingly, PBAF has a higher probability to stably engage chromatin inside hubs indicating that hubs contain a unique nucleosomal scaffold compared to global chromatin. Dual color imaging of PBAF in hubs near H3.3 or HP1α reveals that PBAF targets both euchromatic and heterochromatic regions with distinct genome binding kinetics that mimic chromatin stability. Removal of PBAF’s bromodomains stabilizes H3.3 and HP1α binding within chromatin indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data, suggests that PBAF differentially and dynamically engages a variety of chromatin structures involved in both activation and repression of transcription via bromodomains. Furthermore, PBAF’s binding stability on chromatin may reflect the chromatin remodeling potential of different bound chromatin states.Statement of SignificanceTranscriptional bursting involves a gene rapidly switching between transcriptionally active and inactive states. To regulate transcriptional bursting, chromatin must interchange between euchromatin and heterochromatin to permit or restrict access of transcription factors including RNA Polymerase II to enhancer and gene promoters. However, little is known regarding how chromatin remodelers dynamically read a rapidly changing 4D epigenome. We used live-cell single molecule imaging to characterize the spatiotemporal chromatin binding dynamics of PBAF, a chromatin remodeler that accesses both euchromatin and heterochromatin to regulate transcription. PBAF cycles on and off chromatin hubs in select nuclear regions where it distinctly engages euchromatin and heterochromatin via bromodomains in its BAF180 subunit. Our study provides the framework to understand how the 4D epigenome is regulated.

The p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

More than 50% of cancer patients harbor p53 mutations, highlighting the essential role of p53 in tumor suppression. In response to various stress signals, p53 targets the TFIID-mediated transcription machinery to stimulate expression of vast gene networks involved in diverse cellular pathways. Understanding how p53 turns on TFIID-mediated transcription initiation at the single molecule level will advance our knowledge of p53's tumor suppression activity.Despite 20 years of biochemical studies, it remains elusive how p53 recruits TFIID and other basal transcription factors (e.g. TFIIB and RNA Polymerase II) to facilitate pre-initiation complex formation on various target gene promoters. We hypothesize that p53 induces distinct DNA-binding conformations within basal factors to stimulate assembly culminated in transcription initiation. Therefore, to test our hypothesis, we employed an innovative integrated approach involving single particle cryo-electron microscopy (EM), single molecule fluoresce microscopy and biochemistry.Intriguingly, our studies demonstrated that p53 delivers and promotes stable binding of TFIID to DNA. Unexpectedly, the interaction of TFIID and DNA in turn causes p53 to quickly dissociate from the assembly and promoter. Furthermore, our work suggest that the direct association of p53 and TFIID is sufficient to recruit TFIID onto various target gene promoters. More significantly, p53 induces a common DNA-binding conformation of TFIID. We further identified a novel role of TFIID in anchoring core promoter DNA downstream of the transcription start site.Collectively, these findings indicate that p53, and potentially activators in general, serve as escorts to dynamically recruit and load the basal transcription machinery onto DNA. Importantly, p53 facilitates formation of common DNA-binding forms of basal factors. Overall, our studies are critical for understanding how eukaryotic transcription complexes initiate transcription on different types of protein-coding genes in response to various stress signals.

P53 is a transcriptional activator that binds to its response elements (REs) on target promoters and activates expression of a large number of genes involved in tumor suppression. Previous genome-wide studies showed that p53 binds to its REs in regions densely populated with nucleosomes. However, it is unknown how p53 accesses its target REs in the context of chromatin to regulate transcription.To better decipher the interaction of p53 with chromatin targets, we utilized a combination of in vivo live cell and in vitro single molecule imaging along with bioinformatics and Next Generation Proteomic screens. Our live cell imaging studies indicate a dynamic interaction between p53 and chromatin that varies in distinct sub-nuclear compartments. In vitro, p53 binds a mononucleosome on a native p53 target gene with nearly identical residence times as our live cell measurements. Strikingly, p53 has a higher affinity for its RE when incorporated into nucleosomes compared to an RE on naked DNA, suggesting a novel role for p53 as a pioneer factor. In addition, p53 transiently interacts and alters the structure of nucleosomal DNA, presumably to facilitate access of additional p53 bound chromatin modifiers.Bioinformatic analysis reveal that p53 REs cluster specifically within 2 regions of the nucleosome. Interestingly, p53 interacts with peptides that have strong homology to histones H2A, H2B, and H4 in our next generation proteomic screens. Furthermore based upon crystal structures, these histone peptides are adjacent to the clustered p53 REs, strongly suggesting localization of two physiologically relevant binding platforms for p53 on the nucleosome. Collectively, our studies indicate that p53/histone contacts combined with p53's interaction with REs may enhance p53 directed transcription by creating a stable multivalent platform for p53's recruitment to target promoters.

The vertebrate retina is a distinctly laminar structure. Functionally, the inner plexiform layer, in which bipolar cells synapse onto amacrine and ganglion cells, is subdivided into two sublaminae. Cells that depolarize at light offset ramify in sublamina a; those that depolarize at light onset ramify in sublamina b. The separation of ON and OFF pathways appears to be a fundamental principle of retinal organization that is reflected throughout the entire visual system. We show three clear exceptions to this rule, in which the axons of calbindin-positive ON cone bipolar cells make ribbon synapses as they pass through the OFF layers with three separate cell types: (1) dopaminergic amacrine cells, (2) intrinsically photosensitive ganglion cells, and (3) bistratified diving ganglion cells. The postsynaptic location of the AMPA receptor GluR4 at these sites suggests that ON bipolar cells can make functional synapses as their axons pass through the OFF layers of the inner plexiform layer. These findings resolve a long-standing question regarding the anomalous ON inputs to dopaminergic amacrine cells and suggest that certain ON bipolar cell axons can break the stratification rules of the inner plexiform layer by providing significant synaptic output before their terminal specializations. These outputs are not only to dopaminergic amacrine cells but also to at least two ON ganglion cell types that have dendrites that arborize in sublamina a.

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35Å structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step towards correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a mega-Dalton macromolecular assembly can significantly alter its activity and lead to a TAF4b induced re-programming of promoter specificity.

Cell-type-selective expression of the TFIID subunit TAF II 105 (renamed TAF4b) in the ovary is essential for proper follicle development. Although a multitude of signaling pathways required for folliculogenesis have been identified, downstream transcriptional integrators of these signals remain largely unknown. Here, we show that TAF4b controls the granulosa-cell-specific expression of the proto-oncogene c-jun , and together they regulate transcription of ovary-selective promoters. Instead of using cell-type-specific activators, our findings suggest that the coactivator TAF4b regulates the expression of tissue-specific genes, at least in part, through the cell-type-specific induction of c-jun , a ubiquitous activator. Importantly, the loss of TAF4b in ovarian granulosa cells disrupts cellular morphologies and interactions during follicle growth that likely contribute to the infertility observed in TAF4b-null female mice. These data highlight a mechanism for potentiating tissue-selective functions of the basal transcription machinery and reveal intricate networks of gene expression that orchestrate ovarian-specific functions and cell morphology.

Over 50% of cancer patients harbor a mutation in the tumor suppressor protein p53. In healthy cells during stress, p53 recognizes specific genomic response elements (REs) to induce expression of stress response genes involved in cell cycle arrest, apoptosis and DNA damage. To maintain genomic stability, expression of these tumor suppression genes must be rapidly turned on and off. It is currently poorly understood how p53 spatiotemporally regulates expression of target genes in the complex milleu of the nucleus and how this process goes awry in cancer cells.To better mechanistically decipher changes in stress response gene expression in cancer cells, we established a single molecule imaging system that allows us simultaneously to track p53 and RNA Polymerase II binding along with transcription dynamics in single live cells. Single particle tracking studies indicate a dynamic interaction between p53 and chromatin that varies in distinct sub-nuclear compartments. Live cell monitoring of gene expression from a p53 target gene reveals dynamic bursts of transcription in discrete sub-nuclear regions. Modulation of wild-type p53 levels leads to changes in the transcription burst frequency and intensity. Introduction of cancer associated mutant p53 into cells leads to changes in both p53 sub-nuclear localization and transcription bursting dynamics. Future studies will focus on how chromatin structure dynamically regulates sub-nuclear localization and expression of p53 targets genes. This work is supported in parts via the 4D Nucleome consortia NIH grant 1U01EB021236-01.

A large number of small-sized samples invariably shows that woody species are absent from forest soil seed banks, leading to a large discrepancy with the seedling bank on the forest floor. We ask: 1) Does this conventional sampling strategy limit the detection of seeds of woody species? 2) Are large sample areas and sample sizes needed for higher recovery of seeds of woody species? We collected 100 samples that were 10 cm (length) × 10 cm (width) × 10 cm (depth), referred to as larger number of small-sized samples (LNSS) in a 1 ha forest plot, and placed them to germinate in a greenhouse, and collected 30 samples that were 1 m × 1 m × 10 cm, referred to as small number of large-sized samples (SNLS) and placed them (10 each) in a nearby secondary forest, shrub land and grass land. Only 15.7% of woody plant species of the forest stand were detected by the 100 LNSS, contrasting with 22.9%, 37.3% and 20.5% woody plant species being detected by SNLS in the secondary forest, shrub land and grassland, respectively. The increased number of species vs. sampled areas confirmed power-law relationships for forest stand, the LNSS and SNLS at all three recipient sites. Our results, although based on one forest, indicate that conventional LNSS did not yield a high percentage of detection for woody species, but SNLS strategy yielded a higher percentage of detection for woody species in the seed bank if samples were exposed to a better field germination environment. A 4 m2 minimum sample area derived from power equations is larger than the sampled area in most studies in the literature. Increased sample size also is needed to obtain an increased sample area if the number of samples is to remain relatively low.

Background: The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin. Results: When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity. Conclusions: These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein–protein interactions in cells.

To understand the role of thyroid hormone in metastasis, we studied the expression of the anti-metastatic nm23 gene in eight human hepatocellular carcinoma (HCC) cell lines. These cells differentially expressed the anti-metastatic nm23 gene. A low level of nm23 proteins was found to have a high in vitro invasive activity which correlated closely with an overexpression of the thyroid hormone β1 nuclear receptor (h-TRβ1). Concurrent with the down-regulation of h-TRβ1, the invasive activity of HCC cells was suppressed by the thyroid hormone, 3,3′,5-triiodo- l -thyronine (T3). These results indicate that the invasive activity of HCC cells was regulated by T3, suggesting that T3 could be involved in modulating the functions of nm23.

The tumor suppressor p53 protein possesses activities typical of eukaryotic transcriptional activators; p53 binds to specific DNA sequences and stimulates transcription of the target genes. By a series of deletion and domain-swapping studies, we report here that (i) p53 has two auxiliary domains, which have little effect on the DNA binding activity of its core domain but are capable of modulating its transactivation activity in a target site-dependent manner; (ii) p53 contains two cell-specific transcriptional inhibitory domains, I1 and I2, which are active in Saos-2 and HeLa cells but not in HepG2 and Hep3B cells; and (iii) I1 inhibits the activity of several structurally different activating regions. These results demonstrate that the apparent transcriptional activity of p53 is determined by collaborations among its regulatory domains, its target sites, and the cellular environment.

In humans, transcription Pre-Initiation Complex (PIC) assembly begins with the recognition of the core promoter by the large multi-subunit TFIID complex and culminates with recruitment of RNA Polymerase II. Transcriptional activators, such as the tumor suppressor p53 protein, stimulate transcription initiation, in part by binding sequences upstream of the promoter and help recruit TFIID to core promoter DNA.In response to cellular stress the tumor suppressor p53 protein becomes activated and turns on a variety of target genes in a highly coordinated manner to dictate cellular fate. Cellular levels of p53 are essential in dictating gene expression programs based upon the affinity of p53 for binding sites upstream of the core promoter. Many general transcription factors that bind to the core promoter are known to interact with p53 and potentially alter the affinity of each factor for the promoter DNA. Despite almost 20 years of research, the dynamic interplay between p53 and components within the general transcription machinery at physiological promoters remains poorly understood.We have probed in real-time the association between human p53, TFIID, and various p53 target gene promoters, using a combination of in vitro single molecule tethered particle motion(TPM) and high-resolution TIRF co-localization assays. Our results show that p53 and TFIID act cooperatively to aid in recruitment to promoter DNA. In addition, differences in cooperative assembly of p53 and TFIID on various physiological promoters underscore a mechanism for p53's graded response to cellular stress. TPM analysis reveals p53 and TFIID dependent DNA looping suggesting stable interactions between upstream bound activators and core promoter bound factors. We have further examined the influence of additional general transcription factors on p53 mediated transcription complex assembly.

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, we determined the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their structures as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, our results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation.

p53 plays a central role in tumor suppression. To quickly respond to diverse stress stimuli, p53 binds specific elements in various target promoters to induce vast gene networks for maintaining cellular integrity. p53 stimulates transcription in part by aiding promoter recruitment of the transcription machinery. TFIID, a key component within the transcription machinery, is responsible for binding specific core promoter DNA sequences and recruiting other basal factors including RNA Polymerase II to initiate transcription. However, the exact mechanism underlying how p53 facilitates TFIID-mediated transcription is unclear.Each p53 target gene has a unique arrangement of p53-responsive and core promoter elements. How these various arrangements on different gene promoters regulate the structural architecture of TFIID and the positioning of p53/specific elements remains unknown. Moreover, structural information of p53 bound to its various target promoters and other factors remains elusive. Therefore, we aim to decipher the molecular mechanism underlying p53's ability to stimulate transcription by revealing the biochemical, structural and dynamic basis of TFIID bound to p53 and promoter DNA.To this end, we established unique protein purification strategies to generate high-purity native TFIID complex bound to p53/TFIIA/native promoter DNA. We next determined the 3D structures of TFIID/p53/TFIIA co-complexes on two distinct p53 target gene promoters via single particle cryo-electron microscopy. Strikingly, we discovered a common mode of TFIID binding to different types of promoters. Our biochemical studies showed that p53 significantly promotes TFIID's interaction with DNA. To further mechanistically dissect TFIID's enhanced promoter recognition/binding directed by p53, we examined the dynamic interaction between p53, TFIID and promoter DNA via single molecule TIRF microscopy. Taken together, our structural and functional studies elucidate how p53 facilitates TFIID-mediated transcription initiation complex assembly on different p53 target gene promoters.

The tumor suppressor p53 protein is a transcriptional activator that binds to its response elements (REs) on target promoters and activates expression of a large number of genes involved in tumor suppression. Previous ChIP-seq studies indicate that p53 binds to its REs embedded in regions densely populated with nucleosomes. However, it is unknown if nucleosomes help or hinder binding of p53 to its response elements to regulate transcription.To decipher the interaction of p53 with nucleosomes, we have utilized a combination of Next Generation Proteomics, bioinformatics, bulk biochemical and real-time single molecule FRET assays. Our proteomic assays show that p53 interacts with peptides that have strong homology to histones H2A, H2B, and H4. Biochemical assays indicate that p53 can stably interact with histone peptides in the absence of DNA. Genomic maps of p53 REs and nucleosome positions further reveal that p53 REs cluster specifically within 2 regions of the nucleosome. Furthermore these clustered p53 REs are adjacent to histone regions identified in our proteomic studies, suggesting localization of two physiologically relevant binding platforms for p53 on the nucleosome.We have also established a single molecule FRET assay to characterize dynamic structural changes in nucleosomes with and without p53 REs. Interestingly, our studies reveal that p53 can bind to our biochemically defined nucleosomal surface and dynamically alter the structure of nucleosomal DNA independently of the presence of a p53 RE. Intriguingly, p53 has a higher affinity for its RE when incorporated into nucleosomes assembled with native DNA compared to REs on naked DNA. Thus, our studies indicate that histone/p53 contacts may enhance p53 directed transcription by creating a stable platform for p53 promoter recruitment.

The p53 tumour suppressor protein plays a central role in maintaining genomic integrity in eukaryotic cells. The most significant biological function of p53 is to act as a sequence-specific DNA-binding transcription factor, which can induce the expression of a variety of target genes in response to diverse stress stimuli. The p53 protein contains six highly conserved regions, one of which, termed Box I, is located in the N-terminal transactivation domain (amino acid residues 13 and 26). The second half of the Box I region is crucial for the interaction with the basal transcription machinery and is thus required for p53's activity as a transcription factor. The same region also binds to Mdm2. Since p53 is targeted by Mdm2 for ubiquitin-mediated proteasome-dependent degradation, this region is also essential for the regulation of p53's stability in response to stress signals. Although the first half of Box I is highly conserved, its biological function is not clearly defined. The aim of this study was to characterise this conserved region and investigate its role in the biological functions of p53. We have generated short deletions and point mutations within this region and analysed their effect on p53 function and regulation. Biochemical analyses demonstrate that deletion of residues 13 to 16 significantly increases both the transcriptional transactivation and G(2) arrest-inducing activities of murine p53. Residues 13 to 16 appear to function as a regulatory element in p53, modulating p53-dependent transcriptional transactivation and cell-cycle arrest, possibly by affecting the structural stability of the core domain of the protein. In support of this, the deletion was found to induce second-site reversion of the Val135 temperature-sensitive mutant of murine p53.

Hepatitis B infection is a major causative factor for chronic liver disease and hepatocellular carcinoma. Recent studies have shown RNA interference (RNAi), the process of gene regulation via specific degradation of target mRNA, can be applied to inhibit hepatitis B replication, however administration to patients is a major issue. Short interfering RNA (siRNA) can be encoded as hairpins within DNA, however viral vectors commonly evoke an immune response, limiting the pool of vector available, whilst non-viral vectors are often poorly efficient due to barriers including the nuclear membrane. We were therefore interested to develop the use of polyelectrolyte complexes based on pH responsive cationic polymers containing reducible disulphides to condense siRNA. These vectors are designed to undergo reduction-triggered intracytoplasmic unpacking and release of biologically active siRNA in its final form. The advantage of such a system is the avoidance of the need to translocate the nuclear membrane in non-dividing cells.

Expression of proliferating cell nuclear antigen (PCNA) gene is growth-regulated. The growth dependence of the rat PCNA gene promoter activity was investigated. Cultured cells were transfected with the promoter containing plasmid and recovered for 48 h in serum-free medium to become quiescent. Cells were then cultured in serum-containing medium and harvested at certain intervals after serum-stimulation, and the promoter-directed chloramphenicol acetyltransferase activities in cell extracts were measured. The promoter used in this study contained sequences between -693 and +125 in relation to the transcription initiation site. The promoter activity was found to be serumresponsive. However, the serum-responsiveness of the promoter became less obvious when the amount of the promoter increased; meanwhile, the promoter became active in the control unstimulated (or quiescent) cells. It was suspected that the dosage effect was due to the titration of the negative regulatory factor in quiescent cells. The titration experiment with a reporterless construct as competitor for regulatory factors showed that the excess of promoter molecules reduced the promoter activity in serum-stimulated cells, while causing a transiently increase of promoter activity in quiescent cells. Based on these results, it is postulated that the serum-responsiveness of the rat PCNA promoter is controlled by both negative and positive regulatory factors. Consistent with this proposition, promoter binding proteins of 105 and 114 kDa were identified only in serum-stimulated and quiescent cells, respectively, in addition to several other promoter binding proteins (ranging from 76 to 110 kDa) which were seen in both serum-stimulated and quiescent cells.

A simple scheme was developed to study the mRNA stability of the proliferating cell nuclear antigen (PCNA) gene during cellular transition from the G1/S boundary to a quiescent state. By this scheme, CHO.K1 cells were grown to about 80% confluence and then serum-starved for 40 h for synchronization in a quiescent state. The quiescent cells were serum-stimulated for a period of time (between 8 h and 12 h) and then grown in serum-free medium until being harvested for further analyses. The cellular PCNA mRNA level was analyzed by Northern blotting. As compared with that in cells which were continuously incubated in serum-containing medium, the decline of the mRNA level, after reaching the peak, in these serum-deprived cells was virtually devoid of mRNA synthesis. Thus, this mRNA decay was taken for the measurement of mRNA stability. The advantage of the scheme is that, unlike the treatment of transcription inhibitors, it does not prevent the cells from completing the rest of the cell cycle before returning to the resting state, and so the mRNA stability observed is cell cycle dependent. In contrast with the previous report that the stability of PCNA mRNA in quiescent cells is less by severalfold than that in S phase cells, our study shows that the mRNA stability of PCNA remained constant during the cellular transition from G1/S boundary to quiescent state.