The Transcriptional Regulator CBP Has Defined Spatial Associations within Interphase Nuclei

It is becoming increasingly clear that nuclear macromolecules and macromolecular complexes are compartmentalized through binding interactions into an apparent three-dimensionally ordered structure. This ordering, however, does not appear to be deterministic to the extent that chromatin and nonchromatin structures maintain a strict 3-D arrangement. Rather, spatial ordering within the cell nucleus appears to conform to stochastic rather than deterministic spatial relationships. The stochastic nature of organization becomes particularly problematic when any attempt is made to describe the spatial relationship between proteins involved in the regulation of the genome. The CREB–binding protein (CBP) is one such transcriptional regulator that, when visualised by confocal microscopy, reveals a highly punctate staining pattern comprising several hundred individual foci distributed within the nuclear volume. Markers for euchromatic sequences have similar patterns. Surprisingly, in most cases, the predicted one-to-one relationship between transcription factor and chromatin sequence is not observed. Consequently, to understand whether spatial relationships that are not coincident are nonrandom and potentially biologically important, it is necessary to develop statistical approaches. In this study, we report on the development of such an approach and apply it to understanding the role of CBP in mediating chromatin modification and transcriptional regulation. We have used nearest-neighbor distance measurements and probability analyses to study the spatial relationship between CBP and other nuclear subcompartments enriched in transcription factors, chromatin, and splicing factors. Our results demonstrate that CBP has an order of spatial association with other nuclear subcompartments. We observe closer associations between CBP and RNA polymerase II–enriched foci and SC35 speckles than nascent RNA or specific acetylated histones. Furthermore, we find that CBP has a significantly higher probability of being close to its known in vivo substrate histone H4 lysine 5 compared with the closely related H4 lysine 12. This study demonstrates that complex relationships not described by colocalization exist in the interphase nucleus and can be characterized and quantified. The subnuclear distribution of CBP is difficult to reconcile with a model where chromatin organization is the sole determinant of the nuclear organization of proteins that regulate transcription but is consistent with a close link between spatial associations and nuclear functions

Condensin and Chromatin Mediated X Chromosome Architecture in Caenorhabditis elegans.

Dosage compensation is an essential gene regulatory mechanism that balances X-linked gene expression and X to autosomal expression in organisms that utilize a chromosome-based method of sex determination. In the worm Caenorhabditis elegans dosage compensation is believed to involve two mechanisms. First, upregulation of the X chromosomes occurs in males and hermaphrodites. The mechanism of X upregulation is not know but is believed to balance X-linked gene expression levels to autosomes in males but causes X hyperactivation in hermaphrodites. This necessitates the second mechanism mediated by the activity of the dosage compensation complex (DCC), which downregulates both hermaphrodite X chromosomes by two-fold. The DCC contains a condensin-like complex, condensin IDC, similar to mitotic condensin, suggesting that it may compact the X chromosomes. The goal of my thesis is to address the long-standing hypothesis that DCC activity results in changes in X chromosome structure and explore the highly debated mechanism of X upregulation. In this research, I have uncovered that MYS-1 mediated H4K16ac decondenses the male X chromosome and contributes to upregulation of gene expression on the chromosome. This novel work supports the X upregulation hypothesis. I have also showed that condensin IDC, and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction, providing the first evidence linking condensin-mediated chromosome compaction to chromosome-wide repression of gene expression. My work provided evidence that changes in higher order organization of the X chromosome plays a role both in X upregulation and condensin IDC mediated repression.PhDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133384/1/aclau_1.pd

PML nuclear bodies and the spatial analysis of interphase mammalian cell nuclear architecture

Promyelocytic leukaemia nuclear bodies (PML NBs) are found within the nucleus of mammalian cells. Numbering between 10 and 30 per nucleus, they are an obvious feature of the nuclear landscape, yet their functions have still to be unambiguously defined. In the mammalian nucleus, compartmentalization of functions is apparent, as reflected in the wide-range of other nuclear compartments that can be identified. Quantification of relationships between PML NBs and other nuclear functional compartments is essential for a complete understanding of PML NB function. Initially, PML size, number, distance relationships, and spatial organisation in relation to each other, and the nuclear boundary and centroid, under the spatial point pattern theory hypothesis of Complete Spatial Randomness (CSR), were investigated in both normal and SV40 transformed MRC5 and WI38 human foetal lung fibroblasts. This was also completed in normal MRC5 cells treated with heat shock, and interferon β (both of which alter PML NB morphometrics), and also serum starvation. PML NBs appeared to locate according to CSR with respect to each other, and inter – PML distances were dependent upon median PML NB number per nucleus. PML NBs did not tend to associate with the nuclear centroid, and were repelled from the nuclear boundary in all cell lines and conditions. The distance and spatial organisation relationships between PML NBs and eleven different nuclear compartments were also compared and contrasted in the cell lines and conditions mentioned previously. PML NBs were shown to share strong distance and spatial organisation relationships with the 11S immunoproteasome regulator, SC35 domains, and transcriptional compartments in normal asynchronous nuclei, and with telomeres in transformed cells, highlighting likely functions for the bodies. Lastly, the three dimensional spatial preference of functional compartments in the nucleus was determined using an aggregate map, which provided a novel means to visualise the nuclear location of functional compartments in relation to each other, and under different cellular conditions. Spatial preference fell into four categories: 1) diffuse, 2) annular, 3) core, and 4) polar. Nucleoli and RNA polymerase maintained their spatial preference across cell lines and conditions, whereas other compartments showed altered spatial preferences. Interestingly, viral transformation led to global disorganisation of the nucleus, where most compartments (including PML NBs) reverted to a diffuse spatial preference

Characterization Of Epigenetic Plasticity And Chromatin Dynamics In Cancer Cell Models

Cancer progression is driven by cumulative changes that promote and maintain the malignant phenotype. Epigenetic alterations are central to malignant transformation and to the development of therapy resistance. Changes in DNA methylation, histone acetylation and methylation, noncoding RNA expression and higher-order chromatin structures are epigenetic features of cancer, which are independent of changes in the DNA sequence. Despite the knowledge that these epigenetic alterations disrupt essential pathways that protect cells from uncontrolled growth, how these modifications collectively coordinate cancer gene expression programs remains poorly understood. In this dissertation, I utilize molecular and informatic approaches to define and characterize the genome-wide epigenetic patterns of two important human cancer cell models. I further explore the dynamic alterations of chromatin structure and its interplay with gene regulation in response to therapeutic agents. In the first part of this dissertation, pancreatic ductal adenocarcinoma (PDAC) cell models were used to characterize genome-wide patterns of chromatin structure. The effects of histone acetyltransferase (HAT) inhibitors on chromatin structure patterns were investigated to understand how these potential therapeutics influence the epigenome and gene regulation. Accordingly, HAT inhibitors globally target histone modifications and also impacted specific gene pathways and regulatory domains such as super-enhancers. Overall, the results from this study uncover potential roles for specific epigenomic domains in PDAC cells and demonstrate epigenomic plasticity to HAT inhibitors. In the second part of this dissertation, I investigate the dynamic changes of chromatin structure in response to estrogen signaling over a time-course using Estrogen Receptor (ER) positive breast cancer cell models. Accordingly, I generated genome-wide chromatin contact maps, ER, CTCF and regulatory histone modification profiles and compared and integrated these profiles to determine the temporal patterns of regulatory chromatin compartments. The results reveal that the majority of alterations occur in regions that correspond to active chromatin states, and that dynamic chromatin is linked to genes associated with specific cancer growth and metabolic signaling pathways. To distinguish ER-regulated processes in tamoxifen-sensitive and in tamoxifen-resistant (TAMR) cell models, we determined the corresponding chromatin and gene expression profiles using ER-positive TAMR cancer cell derivatives. Comparison of the patterns revealed characteristic features of estrogen responsiveness and show a global reprogramming of chromatin structure in breast cancer cells with acquired tamoxifen resistance. Taken together, this dissertation reveals novel insight into dynamic epigenomic alterations that occur with extrinsic stimuli and provides insight into mechanisms underlying the therapeutic responses in cancer cells

Nuclear Pore Proteins In Regulation Of Chromatin State And Gene Expression

Nuclear pore complexes are best known for their regulation of nucleocytoplasmic transport as integral components of the eukaryotic nuclear envelope. Over the years, their importance in regulation of genome function has become apparent. Many of the 30 individual nuclear pore proteins, Nups, have been found to play distinct roles interacting with and regulating various genomic targets, especially in a cell-type specific manner. The mechanism behind this regulation is often unknown. We have developed a method by which to study the roles of Nups on chromatin using an ectopic-tethering system. Drosophila melanogaster provide a powerful tool with which to combine many genetic elements of interest together in individual organisms quickly and efficiently, and additionally has allowed for powerful high-resolution visualization of chromatin structure perturbations through the imaging of their larval salivary gland polytene chromosomes. Using this system we observed that tethering Nups to chromatin was sufficient to induce chromatin decondensation, visualized by robust and reproducible loss of DNA and histone fluorescene signal associated with Nup binding. Additionally we observed recruitment of chromatin-remodeling complex PBAP, and reliance on PBAP for the observed Nup-induced decondensation, suggesting an important functional relationship between these proteins. We then took our findings and hypotheses generated from this ectopic-tethering imaging system to next conduct functional biochemical analysis of these proteins in Drosophila S2 cell culture. We found that nucleoporin Elys has a robust biochemical interaction with components of PBAP in an endogenous context, supporting the recruitment of these proteins we observed via immunofluorescence. Additionally, MNase experiments determined that Elys was critical for facilitating the formation and/or maintenance of open chromatin, both genome-wide and on a local nucleosomal level at Elys target genes. Together these results demonstrate the importance of nucleoporins in regulation of chromatin structure, and provide one mechanism to explain this phenomenon. These findings are of particular interest in the fields of chromatin biology and the study of nuclear pore protein function, demonstrating a possible explanation for not only associations of NPCs with decondensed chromatin at the nuclear periphery, but also regulation of Nup target gene expression, through regulation of chromatin accessibility

Transcription silencing and sub-nuclear positioning of the HIV-1 provirus

The human immunodeficiency virus (HIV-1) is a retrovirus that integrates into host cell\u2019s chromatin for gene expression and replication. As integrated provirus HIV-1 is able to persist for long periods of time during antiretroviral therapy in quiescent memory T cells reservoirs. Understanding how these reservoirs are established, maintained, and reactivated is essential for developing methods to target and eventually eradicate HIV-1 infection. Latency is likely established and maintained by numerous blocks at multiple steps in the HIV-1 gene expression pathway, which potentially complicates eradication strategies that aim at the purging of HIV-1 reservoirs from the infected patient. Recently, it has been proposed that the spatial distribution of genes within the nucleus contributes to transcriptional control allowing optimal gene expression as well as constitutive or regulated gene repression. Hence, the position of the provirus within the nucleus and its long-range interaction with other genomic regions could be another unexplored level of HIV-1 transcription control. In order to gain insight in the conformation of chromatin at the site of HIV-1 integration we exploited seven different cell lines carrying a single latent provirus, which represent well-characterized models of HIV-1 latency. In the silenced state, the provirus was consistently found at the nuclear periphery. After induction of transcription the location of the transcribing provirus remained peripheral. Furthermore, in the J-lat A1 cell line, chromatin conformation studies revealed that the proviral vector is associated to a pericentromeric region of chromosome 12 (Ch12q12) located at the periphery of the nucleus. Even though the location of the provirus did not change in transcriptionally active cells, the association between these two loci was lost. These results reveal a new mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and reinforce the notion that gene transcription can occur also at the nuclear periphery

The mechanism by which TCERG1 inhibits the growth arrest activity of C/EBPa

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein α (C/EBPα) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBPα. Additionally, the original clone found to interact with C/EBPα consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBPα mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBPα, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBPα-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBPα-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBPα. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBPα was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBPα, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBPα

Studying and manipulating chromatin motion in mammalian cells

Histone modifications such as methylation and acetylation are known to be key determinants in the regulation of gene expression, but little is known about how higher order chromatin structures, and their spatial organisation in the nucleus, can control gene expression. This remains a key question in addressing the role of spatial organisation in genomic function.If changes in nuclear position have a role in gene expression, chromatin within the cell must be able to move distances that would accommodate this. In the first part of my PhD I investigated the range of chromatin motion in living human cells. E.coli Lac operator arrays inserted into the human genome and visualised using Lac repressor protein fused to GFP, are able to move up to 2-3 pm over the period of two hours, distances greater than previously reported and similar to motion observed in yeast. I have also determined whether the position of a locus is conserved from one cell cycle to the next by following cells through mitosis. From this analysis it was concluded that although some aspects of positioning were conserved, loci position was established anew each cell cycle.Although I have shown that chromatin mobility is quite constrained within the nucleus, proteins associated with chromatin have been shown to be highly mobile. I have investigated the effect of different factors that might affect the mobility of linker histones using fluorescence recovery after photobleaching. I have shown that while Su(Var)3-9, responsible for tri-methylation of Lysine 9 on histone 3, and MeCP2, a DNA methylation binding protein, have no effect on linker histone mobility, the methylation of DNA does. In the absence of DNA methylation, linker histones are more tightly bound to the chromatin fibre.In humans it is well established that chromosomes have a gene-density related radial organisation within the cell nucleus. I have mapped the radial position of mouse chromosomes in ES cells to determine if a similar pattern of organisation exists. My results suggest there may be a loose correlation between chromosome size and position within the mouse genome, but not gene density. Furthermore differentiation iii of mouse ES cells, induced changes in the position of some chromosomes, suggesting that gene expression may have a role in chromosome position.Although correlations in nuclear position and expression have been seen in many model organisms, only in budding yeast has there been direct experimental confirmation that position can control gene expression. To determine directly if nuclear position can regulate gene expression in the mouse I aimed to artificially tether a gene to the edge of the mouse nucleus. Arrays of Lac operator sequences were inserted into or near genes. To tether genes to the nuclear periphery, Lac repressor was fused to the integral membrane proteins emerin or LAP2ß. I have shown that these fusion proteins can transiently anchor transfected Lac-operator containing plasmids to the nuclear periphery of mouse cells and that this silences gene expression from these plasmids. Anchoring of endogenous mouse genes was also investigated

AIRE mõju geeniekspressioonile – transkriptsiooni regulatsiooni mehhanismi uuringud koekultuuri rakkudes

Väitekirja elektrooniline versioon ei sisalda publikatsiooneProbleemi kirjaldus. Immuunsüsteemi põhiline ülesanne on võidelda haigustekitajatega, mis üritavad tungida organismi, et seda kahjustada. Samaväärselt oluline on immuunsüsteemi võime hoiduda kehaomaste või väljastpoolt tulevate kahjutute molekulide vastu suunatud immuunreaktsioonist. Seda nähtust nimetatakse immuuntolerantsuseks. Immuuntolerantsuse kujunemisel on suur roll harknäärmel, mis vastutab kehaomaste valkude suhtes tundlike T-rakkude kõrvaldamise eest. Suuresti sõltub see protsess ühest valgust nimega autoimmuunsuse regulaator (AIRE). AIRE olemasolul avalduvad harknäärmes kehaomased valgud, mis viiakse kokku arenevate T-rakkudega. T-rakk, millel tekib tugev reaktsioon nende valkude suhtes, sureb enne, kui jõuab harknäärmest lahkuda. Kui harknäärmes puudub AIRE, jäävad ohtlikud T-rakud ellu, siirduvad vere- ning lümfiringe kaudu teistesse kudedesse ja vallandavad autoimmuunrünnaku, mis hävitab koe, põhjustades seeläbi raskeid haiguseid. Tulemus ja kasutegur. Käesolevas doktoritöös uurisime, kuidas mõjutab AIRE funktsiooni see, kui valgule lisada biokeemilisel viisil atsetüülrühmasid. Leidsime, et muutuvad AIRE paiknemine rakutuumas, valgu stabiilsus ning võime aktiveerida kehaomaste valkude avaldumist. Lisaks analüüsisime AIRE valgus esinevat mutatsiooni, mis põhjustab inimestel AIRE-puudulikkusest tingitud autoimmuunhaigust. Selgus, et mutatsioon lõhub AIRE valgu struktuuri ning kuigi AIRE on rakus olemas, ei pääse ta rakutuuma, et kehaomaste valkude avaldumist aktiveerida. Doktoritöö viimases osas keskendusime muutustele genoomi struktuuris, mis kaasnevad AIRE poolt aktiveeritud kehaomaste valkude avaldumisega. Avastasime vastupidiselt ootustele, et muutused toimuvad kehaomaste valkude geenidest kaugel olevates genoomiosades, viidates sellele, et AIRE-l on seni teadaolevast palju laialdasem mõju genoomi ülesehitusele. Kokkuvõtvalt, uurides AIRE valku mõjutavaid tegureid ning AIRE enda toimet genoomi struktuurile, suudame paremini mõista immuuntolerantsuse ning autoimmuunhaiguste tekkemehhanisme.Description of the problem. The main function of the immune system is to fight off pathogens that try to invade and harm the body. At the same time, the immune system has to block any immune reactions against harmless antigens stemming from the organism itself or from the environment. This phenomenon is called immune tolerance. The thymus plays a major role in establishing tolerance towards self-antigens by eliminating autoreactive T-cells. This process is primarily controlled by a single protein called autoimmune regulator (AIRE). AIRE promotes the expression of self-antigens that are presented to developing T-cells in the thymus. T-cell that strongly react to the self-antigens, will die before they leave the thymus. If AIRE is absent from the thymus, then the self-reactive T-cells will survive and migrate to other tissues, which can be targeted and destroyed by these T-cells causing an autoimmune disease. Result and benefit. In this thesis, we investigated the effect of acetylation of the AIRE protein. We found that it alters the localisation and stability of the protein and eventually affects the expression of self-antigens. Additionally, we analysed a mutation in the AIRE protein that causes AIRE-deficiency and autoimmunity in humans. We conclude that the mutation destroys AIRE protein structure, and although AIRE is still present in the cells, it cannot move into the nucleus to activate the expression of self-antigens. In the final part of the thesis we explored the changes in the structure of the genome that coincide with the AIRE-dependent activation of self-antigen expression. Contrary to expectations, the genomic alterations occurred far away from self-antigen coding genes suggesting that AIRE has a much broader impact on the gene regulatory processes in the nucleus than previously anticipated. In summary, uncovering factors that affect AIRE function and how AIRE itself contributes to the genomic organisation expand our understanding of the molecular mechanisms behind immune tolerance and autoimmunity