Prothymosin α is a component of a linker histone chaperone

AbstractLinker histone H1 binds with high affinity to naked and nucleosomal DNA in vitro but is rapidly exchanged between chromatin sites in vivo suggesting the involvement of one or more linker histone chaperones. Using permeabilized cells, we demonstrate that the small acidic protein prothymosin α (ProTα) can facilitate H1 displacement from and deposition onto the native chromatin template. Depletion of ProTα levels in vivo by siRNA-mediated mRNA degradation resulted in a decreased rate of exchange of linker histones as assayed by photobleaching techniques. These results indicate that ProTα is a component of a linker histone chaperone

Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals

BACKGROUND: Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator. RESULTS: Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins. CONCLUSIONS: We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals

Overexpression of Prothymosin Alpha Predicts Poor Disease Outcome in Head and Neck Cancer

In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC).Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients.<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard's ratio, HR = 5.2, 95% CI = 2.3–11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage.Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease

Fuzzy complex formation between the intrinsically disordered prothymosin α and the Kelch domain of Keap1 involved in the oxidative stress response.

Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region (38)NANEENGE(45) of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding

Prothymosin a overexpression contributes to the development of pulmonary emphysema

Emphysema is one of the disease conditions that comprise chronic obstructive pulmonary disease. Prothymosin a transgenic mice exhibit an emphysema phenotype, but the pathophysiological role of prothymosin a in emphysema remains unclear. Here we show that prothymosin a contributes to the pathogenesis of emphysema by increasing acetylation of histones and nuclear factor-kappaB, particularly upon cigarette smoke exposure. We find a positive correlation between prothymosin a levels and the severity of emphysema in prothymosin a transgenic mice and emphysema patients. Prothymosin a overexpression increases susceptibility to cigarette smoke-induced emphysema, and cigarette smoke exposure further enhances prothymosin a expression. We show that prothymosin a inhibits the association of histone deacetylases with histones and nuclear factor-kappaB, and that prothymosin a overexpression increases expression of nuclear factor-kappaB-dependent matrix metalloproteinase 2 and matrix metalloproteinase 9, which are found in the lungs of patients with chronic obstructive pulmonary disease. These results demonstrate the clinical relevance of prothymosin a in regulating acetylation events during the pathogenesis of emphysema

Identification of distinct SET/TAF-Iβ domains required for core histone binding and quantitative characterisation of the interaction

<p>Abstract</p> <p>Background</p> <p>The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Iβ belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Iβ, we designed several SET/TAF-Iβ truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays.</p> <p>Results</p> <p>Wild type SET/TAF-Iβ binds to histones H2B and H3 with K_dvalues of 2.87 and 0.15 μM, respectively. The preferential binding of SET/TAF-Iβ to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Iβ, as well as the H3 amino-terminal tail, are dispensable for this interaction.</p> <p>Conclusion</p> <p>This type of analysis allowed us to assess the relative affinities of SET/TAF-Iβ for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Iβ and can be valuable to understand the role of SET/TAF-Iβ in chromatin function.</p

Proteomic Analysis of Pichindé virus

The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-α. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease

Single-Molecule Investigation of Chromatin-Associated Factors in Genome Organization and Epigenetic Maintenance

The central dogma of biology has laid the foundation for understanding gene expression through the mechanisms of transcription and translation. However, another layer of eukaryotic gene regulation lies in the complex structure of chromatin. This scaffold of structural proteins and enzymatic regulators determines what genes are expressed at what times, leading to cell differentiation, cell fate, and often disease. Currently, the field of chromatin biology has relied on basic biochemistry and cellular assays to identify key epigenetic regulators and their role in genomic maintenance. For this thesis work, I have developed a biophysical platform to study chromatin-associated factors at the single-molecule level (Chapter 2). This methodology allows us to extract key mechanistic details often obscured by standard bulk methodologies. Using this platform, we posed the question of how epigenetic factor, Polycomb repressive complex 2 (PRC2) engages with chromatin (Chapter 3). PRC2 is a major epigenetic machinery that maintains transcriptionally silent heterochromatin in the nucleus and plays critical roles in embryonic development and oncogenesis. It is generally thought that PRC2 propagates repressive histone marks by modifying neighboring nucleosomes in a strictly linear progression. However, the behavior of PRC2 on native-like chromatin substrates remains incompletely characterized, making the precise mechanism of PRC2-mediated heterochromatin maintenance elusive. Our understanding of this process was limited by the resolution of structural techniques that fail to identify PRC2-binding modes on long chromatin substrates. In short, we found direct evidence that PRC2 can simultaneously engage nonadjacent nucleosome pairs. The demonstration of PRC2\u27s ability to bridge noncontiguous chromosomal segments furthers our understanding of how Polycomb complexes spread epigenetic modifications and compact chromatin. In addition to this single-molecule chromatin binding technology, I also created a singlemolecule platform harnessing correlative force and fluorescence microscopy to assay the material properties of phase separated condensates (Chapter 2). This assay combined methodology to visualize condensate formation at the single-molecule level, in addition to optical trapping of individual droplets to investigate their material properties. Utilizing this technology, we interrogated the role of linker histone H1 (Chapter 4). The linker histones are the most abundant group of chromatin-binding proteins that bind and organize eukaryotic chromatin. However, roles for the diverse and largely unstructured H1 proteins beyond chromatin compaction remain unclear. We used correlative single-molecule force and fluorescence microscopy to directly visualize the behavior of H1 on DNA under different tensions. Unexpectedly, our results show that H1 preferentially coalesces around nascent, relaxed singlestranded DNA. In vitro bulk assays confirmed that H1 has a higher propensity to form phaseseparated condensates with single-stranded DNA than with double-stranded DNA. Furthermore, we dissected the material properties of different H1:DNA condensates by controlled droplet fusion with optical tweezers, and found that increased DNA length and GC content result in more viscous, gel-like H1 condensates. Overall, our findings suggest a potential role for linker histones to sense and coacervate single-stranded nucleic acids in the nucleus, forming reaction hubs for genome maintenance. This work also provides a new perspective to understand how various H1 subtypes and disease-associated mutations affect chromatin structure and function. In summary, we have gained a greater understanding of the biophysical basis for chromatin regulation by both PRC2 and histone H1. Both of the biophysical platforms created for these studies can be applied to various new targets in chromatin biology. They will enable the investigation of a multiplicity of binding interactions, regulatory mechanisms, and material properties of protein-nucleic acid complexes (Chapters 5 & 6). I believe single-molecule techniques will become a major toolset to study chromatin biology, identifying the intricacies and interactions between epigenetic factors and our genome