Mark T. Groudine - US grants

DESCRIPTION (Adapted from applicant's abstract): The c-myc proto-oncogene, whose expression is linked to growth and differentiation in normal cells, is expressed aberrantly in many neoplastic states, including lymphomas, leukemias and small cell lung carcinomas. Molecular analyses of the regulation of expression of the c-myc gene in normal and neoplastic cells have uncovered novel mechanisms for the transcriptional regulation of eukaryotic gene expression. One level of control of c-myc expression is modulation in the quantity and ratio of transcripts that initiate from the two c-myc promoters, P1 and P2. A second and novel mode of transcriptional control, a block to transcription elongation, is also regulated in normal cells. This mechanism, which was described originally in the human c-myc gene and in the murine c-myc gene, controls the amount of initiated transcription that elongates past a block at the 3' end of exon 1 to produce full length c-myc transcripts. The c-myc elongation block operates in both a promoter-specific and regulatable fashion. Transcription initiated at the P1 promoter constitutively reads through the exon 1 block, whereas transcription for the P2 promoter can be modulated to either read through or be blocked. In normal cells, P2 is the predominant promoter. In contrast, in Burkitts's lymphoma (BL) cells, which are characterized by translocation that juxtapose c-myc and immunoglobulin (Ig) sequences, there is a shift of transcription initiation predominantly to the P1 promoter. Thus, the elongation block is abrogated in BL cells, resulting in high levels and/or constitutive synthesis of c-myc RNA. The investigator working hypothesis is that factors confer termination or antitermination activities to RNA polymerase II complexes in a promoter-specific fashion. These factors modify the polymerase complex to recognize or read through downstream block signals. In normal cells, this association is regulatable at the P2 promoter, where as the complex formed at the P1 promoter confers constitutive read through. In BL cells, the shift to P1 transcription may be due to the presence of trans-acting factors that suppress P2 activity and/or increase P1 utilization, or due to cis effects on promoter utilization and strength imposed by the proximity of the c-myc promoters to Ig sequences as a consequence of the translocation. The goal of this proposal is to identify the promoter-specific elements and trans-factors essential in conferring the read-through and "block" modes to polymerase II complexes transcribing the c-myc gene, and to determine the molecular basis of the abrogation of the c-myc elongation block and constitutive expression of c-myc in BL.

The c-myc proto-oncogene, whose expression is linked to cell growth and differentiation, is expressed aberrantly in many neoplastic states, including lymphomas, leukemias and small cell lung carcinomas. Molecular analyses of the regulation of expression of the c-myc gene in normal and neoplastic cells have uncovered novel mechanisms for the transcriptional regulation of eukaryotic gene expression. One level of control of c-myc expression is modulation in the quantity and ration of transcription initiating from the c-myc P1 and P2 promoters. In addition, a second and novel mode of transcription regulation, a block to transcription elongation, is also regulated in normal cells. This mechanism, which is promoter-specific and regulatable, controls the amount of initiated transcription that elongates past a block at the 3' end of exon 1 to produce full length c-myc transcripts. Transcription initiated at the P1 promoter reads through the exon 1 block, whereas transcription from the P2 promoters can be modulated to read through or block. In normal cells, P2 is the predominant promoter. In Burkitt's lymphoma (BL) cells, characterized by translocations that juxtapose c- myc and immunoglobulin (Ig)sequences, the non-translocated c-myc gene is unexpressed. However, in those translocations in which the first exon of c-myc is intact, there is a shift of transcription initiation predominantly to the P1 promoter of the translocated c-myc gene, and the elongation block is abrogated, resulting in high or constitutive levels of steady-state c-myc RNA. In BL cells, the shift to P1 transcription may be due to the presence of trans-acting factors that suppress P2 activity and/or increase P1 utilization, or due to cis effects on promoter utilization and strength imposed by the proximity of the c-myc promoters to Ig regulatory sequences as a consequence of the translocation. The goals of this proposal are to determine the molecular basis of the abrogation of the c-myc elongation block and constitutive expression of c-myc in BL. In addition, we will attempt to identify elements within the Ig locus that are postulated to maintain the translocated c-myc gene in an active state in cells in which the normal c-myc allele is no longer active. We will also test the hypothesis that preferential use of the P1 promoter and consequent read- through transcription of the intact c-myc gene are essential steps in the neoplastic transformation of B cells.

Treatment of human tumors with ionizing radiation has been an effective treatment of cancer for over 50 years. However, the continued limitation of this therapy is the inherent radiation resistance of some tumor types compared with others. The molecular basis of this difference in resistance is unknown. Throughout evolution, organisms have evolved a conserved mechanism of resistance. Induction of DNA damage signals a surveillance mechanism which arrests cells in the G2 phase of the cell cycle before entering mitosis where residual DNA breaks result in chromosome aberrations. During arrest in G2, DNA breaks are repaired and when repair is completed, the cells resume cycling. Two of the genes, Rad9 and Mec1, which control this arrest have been isolated in the budding yeast S. cerevisiae. This group of genes has been named checkpoints. We propose to isolate human homologues of these checkpoint genes and study their role radiation resistance. A simple genetic strategy has been developed to select for inhuman clones able to complement mutations in the yeast checkpoint genes. Human cDNAs which are obtained will be expressed in radiosensitive mammalian cell lines and increases in resistance determined. A survey of expression at the RNA and protein level in clinical specimens from categories of radiation sensitive and resistant tumors will be undertaken. In addition, a method to select for cDNA clones which interfere with Rad9 function in yeast has been developed. The selection will be used onto screen mutagenized Rad9 cDNAs and human cDNA libraries for dominant negative clones of Rad9. These clones will be expressed in mammalian cells and increases in radiation sensitivity monitored. cDNAs which can decrease the radiation resistance of tumors would be an unique resource and have potential clinical benefit in the future.

The human beta-globin Locus Control Region (LCR) is composed of 5 DNase I hypersensitive sites (HS) located 6-22 kb upstream to the epsilon- globin gene on chromosome 11. Naturally occurring deletions which remove the LCR result in the failure to activate all cis linked globin genes. Constructs containing all or some of these HS have been shown to produce high level erythroid specific expression in transgenic mice and transfected cells. Although the LCR contains several regulatory elements, including an erythroid specific enhancer and a nuclear matrix attachment region (MAR), how each element functions to produce a transcriptionally active, DNase I sensitive, early replicating domain in vivo is not understood. In this application, we propose to dissect the role of individual elements of the LCR in the formation of an active, early replicating and transcriptionally active chromatin domain through experiments using homologous recombination to site specifically add back or delete elements of the LCR in their normal chromosomal location. Although homologous recombination has been used to alter structural genes, this approach has not yet been used to study the function of genetic regulatory elements. Through the use of homologous recombination, we will alter regulatory elements in their native chromosomal location and determine the effects of these alterations on both the initiation and maintenance of replication timing, chromatin structure and transcriptional activity. Thus, our proposed experiments represent a novel approach to understanding the contribution of specific control elements to gene structure and function. In addition, we will also use the more conventional transgenic and transfection approaches to complement the homologous recombination analyses. Our specific aims include: (1) accurate mapping of the human beta-globin domain DNase I sensitive and replication domains; (2) determining if and which MAR/enhancer combinations generate a red-cell specific LCR, as measured in transgenic and transfection assays; (3)determining the structural and functional consequences of LCR element additions or deletions in their native chromosomal location by performing homologous recombination experiments to alter mutant and normal beta-globin loci in committed mouse erythroid (MEL) cell hybrids; (4) determining the role of individual elements in the initiation of an active beta-globin domain by performing homologous recombination experiments in non-erythroid cells prior to fusion with Mel cells or in embryonic stem (ES) cells; and (5) determining the role of the LCR in establishing an early replicating, active chromatin structure by analyzing the chromatin structure and replication of mouse DNA at sites of integration of LCR cassettes in transfected MEL cells.

The MyoD cDNA, when expressed under the control of a viral LTR and transfected into a variety of fibroblast and adipoblast cell lines, converts these cells to muscle. While not yet proven MyoD acts as if it were a master regulatory gene for myogenesis. The MyoD protein is nuclear and preliminary work suggests it binds DNA. The present proposal is to: (1) Characterize MyoD using detailed mutagenic analysis. (2) Study its binding to specific DNA sequences; study its interaction with other cellular proteins. (3) Explore preliminary indications that MyoD positively activates its own synthesis but negatively activates its mRNA utilization.

Treatment of human tumors with ionizing radiation has been an effective treatment of cancer for over 50 years. However, the continued limitation of this therapy is the inherent radiation resistance of some tumor types compared with others. The molecular basis of this difference in resistance is unknown. Throughout evolution, organisms have evolved a conserved mechanism of resistance. Induction of DNA damage signals a surveillance mechanism which arrests cells in the G2 phase of the cell cycle before entering mitosis where residual DNA breaks result in chromosome aberrations. During arrest in G2, DNA breaks are repaired and when repair is completed, the cells resume cycling. Two of the genes, Rad9 and Mec1, which control this arrest have been isolated in the budding yeast S. cerevisiae. This group of genes has been named checkpoints. We propose to isolate human homologues of these checkpoint genes and study their role radiation resistance. A simple genetic strategy has been developed to select for inhuman clones able to complement mutations in the yeast checkpoint genes. Human cDNAs which are obtained will be expressed in radiosensitive mammalian cell lines and increases in resistance determined. A survey of expression at the RNA and protein level in clinical specimens from categories of radiation sensitive and resistant tumors will be undertaken. In addition, a method to select for cDNA clones which interfere with Rad9 function in yeast has been developed. The selection will be used onto screen mutagenized Rad9 cDNAs and human cDNA libraries for dominant negative clones of Rad9. These clones will be expressed in mammalian cells and increases in radiation sensitivity monitored. cDNAs which can decrease the radiation resistance of tumors would be an unique resource and have potential clinical benefit in the future.

DESCRIPTION (Adapted from Applicant's Abstract): Expression of the c-myc proto-oncogene is linked to proliferation ad differentiation in normal cells, and aberrant expression of c-myc is characteristic of many neoplastic states, including lymphomas, leukemias, and small cell lung carcinomas. Work in Dr. Groudine's laboratory uncovered a novel mode of eukaryotic transcriptional control, a conditional block to transcription elongation. This mechanism controls the amount of initiated transcripts that elongate past sites of arrest in exon 1 to produce full length c-myc transcripts. During the past granting period, Dr. Groudine and his colleagues have shown that, in mammalian cells, the primary site of polymerase arrest in the c-myc gene and other model templates occurs in the promoter-proximal region (+30 from the start site), and that the simple combination of proximal upstream activators and core promoter elements are sufficient to generate pausing at this site. Elongation of c-myc transcription is regulated in normal cells, and Dr. Groudine's working hypothesis is that two distinct types of RNA polymerase II elongation complexes, which differ in their ability to read through downstream arrest signals, are assembled at the c-myc promoter. Evidence suggests that transcriptional activators and enhancers can alter gene expression, at least in part, by influencing the elongation stage of transcription; they appear, therefore, to be capable of enhancing formation of elongation-efficient elongation complexes. In human Burkett's lymphoma (BL) and murine plasmacytoma cells, which are characterized by translocations that juxtapose c-myc and immunoglobulin (ig) sequences, the c-myc elongation block is abrogated, resulting in constitutive synthesis of c-myc RNA. Dr. Groudine and his colleagues have discovered a novel locus control region (LCR) in the Ig heavy chain locus, which becomes linked in cis to c-myc as a consequence of these translocations. Preliminary functional analysis of this element supports a model in which deregulation of c-myc in BL and plasmacytomas results from cis effects imposed on c-myc by this LCR. The goals of this proposal are to determine the molecular basis of promoter-proximal pausing and the composition of RNA polymerase II transcription complexes that differ in elongation efficiency, as well as to further elucidate the mechanisms by which the newly discovered 3'Calpha IgH LCR deregulates c-myc expression. Specifically, Dr. Groudine proposes to: (1) determine the role of the transcribed template, including its chromatin structure, in promoter proximal pausing; (2) determine the subunit composition of elongation efficient and elongation deficient transcription complexes, and identify and isolate factors that affect transcriptional elongation; (3) determine the cis components of the 3' Calpha LCR involved in the deregulation of c-myc expression in cell culture models, and isolate factors that interact with these sequences; and (4) determine the function of the 3' Calpha LCR in vivo by transgenic analyses of c-myc and reporter genes linked to the LCR, and in its native chromosomal position using novel homologous recombination strategies.

We propose to develop a mouse model of human sickle cell anemia which faithfully reproduces the clinical features of this disease. These include anemia, hemolysis, sickling crises with infarction and end-organ damage involving the spleen, kidneys, lungs, liver, eyes, and central nervous system. We will use established procedures for generating transgenic mice to introduce human alpha and beta-S globin genes. Our approach will include the introduction of a yeast artificial chromosome (YAC) containing the whole beta-globin locus in which the beta-A gene is replaced by the beta-S gene, as well as human alpha globin genes that are expressed in a stage-specific manner. We will combine this approach with the inactivation of the endogenous murine beta globin locus and alpha globin genes by homologous recombination in embryonic stem (BS) cells. The inactivation of these genes will be accomplished by a combination of approaches, including targeted mutagenesis of the mouse beta-globin locus control region (LCR) and the alpha-globin upstream control element, as well as deletion of the structural genes. Adult mice derived from cross- breeding these lines will express hemoglobin S exclusively, reproducing the pattern of globin gene expression in SS patients. The generation of this murine model of sickle cell disease is essential for testing the therapeutic efficacy of the different strategies for gene therapy proposed in this program project grant. In addition, the model will be useful for studies of the pathophysiology of sickle cells, and for testing the effects of pharmacologic agents that may ameliorate clinical symptoms.

DESCRIPTION: (Adapted from investigator s abstract) In a variety of species, the activity of the b-globin locus is regulated by a locus control region (LCR). The human and murine LCRs are comprised of five DNase I hypersensitive sites (5' HS 1-5) located 5' prime of the embryonic beta-like globin genes. Studies conducted by the P.I. indicate that naturally occurring deletions of the human LCR results in a failure to activate the beta-globin locus at the levels of chromatin transcription and replication timing. In addition, this group has shown that the LCR is essential for the function of the origin of replication of the locus located 50 kb upstream of the LCR, between the delta-and the beta-globin genes. In order to examine how the components of the LCR interact to regulate the endogenous beta-globin locus, homologous recombination (HR) strategies to mutate the endogenous human beta-globin LCR in situ, in somatic cell lines and in murine beta-globin LCR embryonic stem (ES) cells have been developed. Initial analysis of mice derived from the ES cells containing HR-generated mutations of the 5' HS-2 and 5' HS-3 have revealed that the beta-globin locus functions almost normally without these sites, results that would have not been predicted from results of transfection and transgenic assays. Accordingly, the goal of the current proposal is to use the newly developed HR strategies to elucidate how the beta-globin LCR and its component hypersensitive sites regulate the chromium structure, replication timing, and origin use, and transcription of the beta-globin locus. Specifically, this proposal will : 1) Determine the sequence of the human beta-globin LCR that are necessary and sufficient for the formation of the active chromatin structure, transcription, and replication timing and origin use characteristic of the beta-globin locus in the erythroid cells. 2) Determine the sequences necessary and sufficient for replication initiation between the human delta- and beta-globin genes, and determine the mechanism by which the LCR controls origin choice. 3) Determine the consequence of HR-generated LCR mutations on transcription, chromatin structure, and replication of the murine beta-globin locus. These studies will complement and extend previous analysis of the human beta-globin LCR, by determining the consequences of LCR mutations after passage through the germ line and at all stages of erythropoiesis. 4) Optimize the HR analysis of the murine beta-globin LCR by developing strategies to (a) increase the efficacy of generating HRs, and (b) allow rapid analysis of mutation in vitro in chimeric embryonic and adult mice.

The Red Cell Gordon Conference has provided an informal atmosphere for in depth discussions related to the cell and molecular biology of hematopoiesis and clinically related issues. The meeting draws leading scientists and clinicians from around the world. The 1997 meeting should be especially noteworthy, given the major recent advances in several areas. Eight individual sessions are planned, each featuring an introductory talk, three full lectures and a selection of short talks in the following areas: 1. Structure, function and interaction of red cell membrane proteins. 2. Expression and function of erythrocyte proteins in non-erythroid cells. 3. Hematopoiesis I: Origin of hematopoietic stem cells. 4. Hematopoiesis II: Hematopoietic lineage commitment. 5. Signal transduction in hematopoietic cells. 6. Control of red cell gene expression l: Overcoming repressive effects of chromatin. 7. Control of red cell gene expression Il: Control of globin gene expression. 8. Red cell diseases. Two afternoon poster sessions will feature a mixture of presentations from all disciplines. Ten individuals with the most exciting posters will be invited to give a 15 minute presentation on the final evening (a very popular format which was adopted several years ago instead of having a keynote lecture). The diversity of interests and richness of discussions in previous Red Cell Gordon Conferences has led to numerous scientific collaborations and fostered new developments in these important areas of basic and clinical research.

DESCRIPTION (Adapted from applicant's abstract) Regulation of the human beta-like globin genes has been intensively investigated because manipulation of the expression of these genes may permit amelioration of sickle cell disease as well as the beta-thalassemias. The human beta-globin locus is also a classic model of tissue specific gene regulation as well as the developmental regulation of a multigene locus. In recent years there has been a consensus that enhancers upstream of the genes (in the locus control region or LCR) directly interact with the genes and increase their transcription. Using a novel system of reporter gene analysis, however, the applicants have recently demonstrated that a key component of the LCR (5'HS2), as well as other enhancers, does not increase the expression level of a reporter gene to any significant extent, but instead counteracts silencing by repressive chromatin. The investigators have shown that promoter elements can counteract silencing as well as increase transcription; they have also found that characteristics of the integration site strongly influence expression level. The applicants propose that cis-acting transcriptional elements serve primarily to ensure that a gene is expressed in a given lineage, while transcription rate is the product of local chromatin structure and modulation by promoter elements. They also propose a series of experiments to test this hypothesis. The investigators will ask if addition of an enhancer to a locus will stabilize expression; if the proximity of promoter elements to the initiator determines their ability to regulate transcription rate; and if insulators, MARs, and enhancers can alter position effects on expression level. Applicants also will study the coordinate regulation of a model two-gene locus they have developed. All of these experiments will be done in K562 erythroleukemia cells, but they plan to also make ES-derived mice to ask if the 5'HS2 globin enhancer can suppress silencing of gene expression in erythroid cells, and if it can silence expression in non-erythroid cells. These studies may prove useful in the design of gene replacement strategies or drug therapies for the hemoglobinopathies, as well as improving basic knowledge of gene expression.

Globin genes are expressed exclusively in the erythroid lineage, and at distinct stages of development. The mechanisms by which the globin genes are regulated are of interest because a more detailed knowledge of the process is likely to contribute to therapies for severe hemoglobinopathies such as thalassemia and sickle cell disease, and because the globin genes are classic models of complex multigene regulation. Our long-term goal is to understand how the globin genes are expressed only in red blood cells, and how the individual genes are controlled through development. During erythroid differentiation, there is a progressive condensation of chromatin in the nucleus culminating in complete cessation of transcription; the globin genes continue to be transcribed until late in this process. We study a nuclear factor, GATA- 1, that appears to have a central role in the expression of globins and other typically erythroid genes in the terminal phase of erythroid differentiation at all stages of development. We have developed a model in which GATA-1 serves to keep globins and other erythroid genes in an active state while genes not essential for the erythroid program are repressed. To test this hypothesis we have developed an assay that uses the beta-geo reporter to distinguish effects on the level of gene expression from effects on epigenetic stability; previous studies of gene regulation have not separated these two parameters. We propose to investigate the interaction of erythroid transcriptional control elements with chromatin, using the beta-geo assay in conjunction with site- specific recombination to control for position effects. We will dissect the gamma-globin promoter, asking which elements within it regulate the level of gene expression and which counteract repression. We will continue our study of the 5'HS2 globin enhancer, in which we have shown that it acts primarily to relieve repression, and focus on the contribution of its component elements to its function. We will also analyze the role of the chromatin factor HMG-l in developmental regulation of the gamma-globin gene, and the expression of repressive chromatin proteins in the terminal phase. Ultimately we hope that these studies will lead to better understanding of developmental regulation of the globin genes and improved therapies for the hemoglobinopathies..

[unreadable] DESCRIPTION (provided by applicant): We hypothesize that the transcriptional activation and silencing that occur during red cell differentiation are based in epigenetic processes such as chromatin structure, CpG methylation and nuclear positioning. Since these events affect large genomic regions, we hypothesize that co-regulated genes are not positioned randomly on the chromosome or in the interphase nucleus. We propose three aims, combining single locus and genomic approaches, to address these hypotheses experimentally. First, we will study in detail a genomic integration site that displays a transcriptional phenotype reminiscent of "cellular memory" in Drosophila. We will map the chromosomal determinants of this site (chromatin, CpG methylation, nuclear organization) and define the hallmarks of the memory phenotype. We also propose to identify the putative cis-element that confers the cellular memory phenotype and to dissect the regulatory components of this element by targeted modification. In the second aim, we will examine, at the single allele level, the relationship between the positioning of a specific genomic locus (beta-globin) relative to its chromosome territory and establishing/maintaining the tissue-specific transcription state. We also propose to identify the distinct subnuclear compartments to which the globin locus may be directed in different cell types and investigate the role of cis-regulatory sequences and transcription in the positioning of a gene locus in these compartments. In the third aim, we propose a bioinformatics approach to determine the relationship between expression status and genomic distribution of genes that are differentially expressed during hematopoiesis. We will also determine the nuclear organization of active and inactive chromatin compartments during erythropoiesis by measuring chromatin structure genome-wide using a microarray approach and a novel fluorescence in situ hybridization (FISH) technique with immunoprecipitated chromatin as probe. The results of the chromosome distribution, expression and chromatin analyses will be combined with an analysis of the nuclear positioning of identified groups of co-regulated genes during erythropoiesis. We believe these experiments will reveal the epigenetic mechanisms by which erythroid-specific gene expression is achieved and maintained and how these mechanisms have shaped the genomic organization required for the concerted regulation of gene expression in the red cell lineage. [unreadable] [unreadable] [unreadable]

erythroid stem cell; laboratory mouse

[unreadable] DESCRIPTION (provided by applicant): Our genetic, molecular and biochemical analyses of ?-globin gene regulation have yielded novel and surprising results, including that the locus control region (LCR) is not required to initiate or propagate the "open" chromatin structure of the locus or confer stage-specific expression of the ?-like globin genes. In its native location, the primary role of the LCR is to enhance the transition from basal to activated transcription. We now propose to gain further insights into the molecular basis for initiating and propagating the chromatin and transcription states of the ?-globin locus during erythropoiesis. Our Specific Aims include: 1. Define epigenetic features including histone modifications, sequence co-localization, DNA methylation and candidate trans-acting factors involved in the initiation and propagation of active and silent states of the ?-globin locus. We will test the hypothesis that the ?-globin "domain" is larger and more dynamic than formerly appreciated, and that specific sequences, factors and epigenetic modifications are involved in the initiation and propagation of active and silent states of the locus. Initial studies, in collaboration with Dr. X-D Fu (UCSD) who developed the highly sensitive DNA Selection and Ligation (DSL) array-based method, will focus on a 1mb region of human chromosome 11 (h11) centered on the LCR, using a currently available array. We will design and build a similar array for analysis of the mouse locus. 2. Determine the role of cis- acting regulatory elements, transcription and cellular background on domains of histone modifications, factor binding and sequence co-localization in the ?-globin locus. We will use targeted mutation analyses in ES cell derived mice and our h11 transfer system to investigate several hypotheses regarding the regulation of p-globin gene expression. The effects of mutations on the epigenetic states of the locus, including factor binding, histone modifications and sequence interactions, will be determined by DSL analyses in stage-specific erythroid cells from mutant mice and in cell lines containing WT and modified h11s passed through different cellular backgrounds. Correlation of these results with activation state defined by primary transcript FISH, HS formation and the degree and extent of generalized DNase sensitivity will address several models of p-globin gene regulation. 3. Identify novel genes involved in erythroid maturation and p-globin gene expression. We will perform unbiased, genome-wide siRNA screens in the well- characterized G1E-ER cells and in ES derived erythroid progenitor (ES-EP) lines to identify novel factors involved in regulating p-globin gene expression and erythroid maturation. The screen makes use of a lentiviral siRNA library comprised of a redundant set of siRNAs for each known mouse gene. We propose several strategies for the validation, identification and prioritization of candidate siRNAs, as well as for the biochemical and functional analysis of genes identified. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-CA-101 The Role of Cellular Architecture in Normal and Tumor Cell Biology. The cell nucleus is a highly ordered organelle. Gene loci and proteins are dynamically localized to compartments within the nucleus, and compartmentalization is thought to regulate transcription and promote nuclear physiology. However, there also may be negative consequences to this organization. In stimulated B cells, the IgH and c-Myc loci often colocalize in shared transcription factories (sites of concentrated active RNA polymerase II), and IgH and c-Myc are frequent translocation partners in plasmacytomas and Burkitt lymphoma. This correlation suggests that the physical proximity of these two genes within transcription factories leads to the high frequency of chromosomal translocations. To determine the mechanism by which genes initially associate with transcription factories and to test the hypothesis that colocalization of the loci is a causative factor in generating IgH/c-Myc translocations, the IgH and c-Myc loci will be imaged in live cells using 4D (3 dimensions over time) fluorescence microscopy. A major challenge in the field of nuclear structure and function has been to uncover the mechanisms underlying the establishment of nuclear organization and to discover its role in physiology. Contributing to the challenge has been a lack of real time assays in live cells, and in the context of native states of development and disease. Instead, the mechanisms underlying the establishment of order in the nucleus and the contribution of nuclear organization to normal and disease physiology have been inferred from static data points thus leading to multiple interpretations of the data and unresolved hypotheses. Here, we will use 4D fluorescence microscopy to address this challenge. Specifically, we will test the mutually exclusive hypotheses that (1) genes move to pre-existing transcription factories, including IgH during pro-B-cell to pre-B cell maturation and c-Myc to transcription factories pre-occupied by IgH during [unreadable]CD40/IL-4 stimulation and (2) that transcription factories nucleate de novo on genes. 4D microscopy will enable us to distinguish these models by direct visualization in the same experiment. Further, the distance between the IgH and c-Myc loci will be measured through lymphopoiesis and used to determine if the distance between the two loci prior to B cell stimulation influences the probability of their colocalization to the same transcription factory. The c-Myc locus will then be tethered to nuclear pore complexes to forcibly separate the c-Myc locus from the IgH locus and to determine if this nuclear compartment is permissible to transcription. Finally we will determine if forced separation of the IgH and c-Myc loci decreases the frequency of IgH/c-Myc translocations and/or increases the frequency of IgH translocations to other loci. Our general strategy will be to create a line of murine ES cells expressing fluorescently tagged proteins from a polycistonic cassette. The fluorescent protein fusions will label key nuclear compartments and specific loci (via fluorescently tagged LacI and TetR binding to arrays of LacO and TetO binding sites). LacO and TetO arrays will be integrated at the c-Myc and IgH loci, respectively to label those genes. The modified ES cells will then be differentiated into B-cells and imaged using fluorescence microscopy to test the above hypotheses. Notably, this project has been designed so that a single cell line will be used under different experimental conditions, thereby increasing the speed and efficiency of data production. While we will test significant mechanistic hypotheses in the work proposed here, there are many additional questions beyond the scope of this 2-year proposal that can be addressed using these same tools. Thus this project will provide a foundation for long-term studies. Finally, as stimulating the American economy is an objective for this RFA, we will create multiple jobs, purchase several pieces of equipment, support core services at the Fred Hutchinson Cancer Center, and employ the services of external biotechnology companies. PUBLIC HEALTH RELEVANCE: The relationship between the position of gene loci in the nucleus and cell physiology has emerged as an important facet of cell biology. Chromosomal translocations occur when double stranded breaks in DNA are repaired by incorrectly fusing one end of the break to a break in another chromosome, often leading to malignant transformation. It has been proposed that colocalization of gene loci into shared compartments predisposes them to translocations. Here we will test those predictions in the context of Burkitt Lymphoma using the common translocation partners IgH and c-Myc. These two gene loci also frequently colocalize in so- called transcription factories. We will first determine the mechanism by which these gene loci interact with transcription factories and then determine if forcibly separating the two loci decreases the frequency of their translocations.

DESCRIPTION (provided by applicant): Our molecular, biochemical, genetic and microscopic analyses of differential gene regulation during hematopoiesis have yielded novel and surprising results. For example, we made the unanticipated finding that, contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells, ?- globin gene expression initiates at the nuclear periphery prior to relocalization of the gene more centrally where high level expression occurs. Moreover, we showed that the ?-globin locus control region (LCR) is necessary for this relocation. Our work also revealed genes that are co-regulated in the erythroid or myeloid lineages tend to be clustered in the genome, and that in each lineage, distinct chromosomes tend to associate on the basis of the chromosomal distribution of co-regulated genes. We also discovered that MLL5, a member of the mammalian Trithorax group of proteins, is essential for erythroid differentiation in an in vitro model. We now propose experiments using single locus, genomic, proteomic, genetic and high-resolution microscopy approaches to investigate the relationships among nuclear localization, initiation and maintenance of transcription state, and the mechanism by which MLL5 regulates erythroid differentiation. Specifically, we propose to: 1. Determine the relationships between peripheral localization and transcriptional activity of gene loci during differentiation. To accomplish this, we will use a combination of high-resolution microscopy, mutagenesis of the native ?-globin locus, and tethering of wildtype (WT) and mutant loci to nuclear pore complexes (NPC) and lamina during murine erythropoiesis. We will also determine the genome-wide alterations in peripheral localization and expression state during erythroid differentiation. 2. Determine the molecular basis of cellular memory. We will use the mouse Toll like receptor 4 (Tlr4) model of monoallelic expression to identify cis-sequences and trans-factors that specify positioning of the inactive and active alleles in different nuclear compartments. Using live cell imaging, we will test the hypothesis that maintenance of the alleles in distinct compartments is involved in propagation of transcription state in the absence of the signals responsible for establishing that state, so called "cellular memory". 3. Determine the composition and function of MLL5-containing complexes during differentiation. To test the hypothesis that MLL5 functions are mediated via interactions with different protein partners, we will identify MLL5-interacting proteins during erythroid differentiation in vitro and in vivo, and, using genomic approaches, determine the function of MLL5 containing complexes during erythropoiesis. We also will perform genetic and biochemical screens in Drosophila melanogaster to identify evolutionarily conserved dMLL5 interacting proteins and regulated pathways to complement and inform our analysis of MLL5 functions in mice. In combination, these experiments will lead to a greater understanding of gene activation and silencing in the hematopoietic lineage. PUBLIC HEALTH RELEVANCE: Localization of genes in different compartments the nucleus is a mechanism by which genes are activated and silenced, and various cellular processes, including differentiation, are regulated. Importantly, several malignancies, including Burkitt lymphoma, acute myeloid leukemia (AML) and breast cancer are thought to arise from alterations in nuclear organization. These data highlight the importance of understanding the relationships between gene regulation and nuclear organization.

DESCRIPTION (provided by applicant): The goal of this project is to produce comprehensive, high-definition maps of mouse regulatory DNA marked by DNaseI hypersensitive sites to parallel the human catalogue currently under production by the ENCODE Project. Digital DNaseI technology enables efficient genome-wide mapping of accessible chromatin and DNaseI hypersensitive sites. The core regions of DNaseI hypersensitive sites are constitutively populated by regulatory factor binding sites, the nucleotide-resolution footprints of which may be systematically exposed on a genome-wide scale by ultra-deep sequencing. DNaseI hypersensitive sites exhibit marked cell-type variability;accordingly, production of a comprehensive catalog will require surveying a wide range of cell types. Cell types targeted under this proposal include murine analogues of the ENCODE Tier 1 and Tier 2 common reference cell lines;a broad spectrum of primary adult tissues;embryonic stem cells;and sentinel tissues amenable to sequential temporal profiling during development. The production of a parallel, high-quality, high-resolution compendium of mouse regulatory DNA will greatly enhance the value of the human ENCODE project and will provide a rich independent and unique resource for evolutionary, functional, and model organism genomics. PUBLIC HEALTH RELEVANCE: Relevance to Public Health Understanding the genetic basis of human disease requires detailed knowledge of the functional elements of the human genome which may be subject to polymorphism. The ENCODE Project seeks to identify all of the functional elements in the human genome, and present project seeks to greatly increase the value of the ENCODE data by providing a parallel catalogue of regulatory DNA in the mouse genome. This project is therefore expected to provide key insights into the importance of elements in the human genome, and to provide an unprecedented resource for rational functional modeling of human disease in the mouse.

DESCRIPTION (provided by applicant):This application proposes to create a new energy-efficient Data Center that will house, power, cool, network, and safeguard the rapidly expanding high-performance computing and data storage systems that are essential to the research of Fred Hutchinson Cancer Research Center (servers and storage hardware are not part of this proposal). The proposed Data Center project is a critical renovation that will improve research productivity in virtually all research programs at the Hutchinson Center, an institution with more than 2800 employees, including 200 faculty and 400 trainees, who work to understand, treat, and prevent cancer and other human diseases. We are rapidly reaching a point when the burgeoning amount of research data generated by our scientists -- particularly those involved in data-intensive projects such as cancer genomics, genome-wide association studies, the application of proteomics to cell biology and discovery of early biomarkers of cancer, and mathematical modeling of epidemics and protein structure -- will exceed our capacity to cool and safeguard that data. To eliminate severe IT-infrastructure constraints on research, we propose a major alteration of 4,806 gsf to create a 389-kW consolidated Data Center. The new facility will increase the capacity of the Hutchinson Center data-center core facilities by more than 50% and consolidate six of nine current data- center spaces into a single Data Center. Modular design will permit expansion of data handling capacity to meet projected needs into 2017. Consolidation will greatly improve energy efficiency, operational efficiency and our ability to monitor the safety of research data. The state-of-the-art hot-air/cold-air separation design for the project maximizes cooling capacity, a current limitation in data-center capacity. The design also takes advantage of local temperate climate for additional energy savings. Auxiliary goals for the new Data Center are better space utilization and more efficient operational management. Furthermore, the new Data Center will enable disaster recovery of 1 Petabyte of data, which we project for FY2013, by creating a redundant storage infrastructure. The resulting increases in research capacity, throughput, and energy-efficiency provided by new Data Center will catalyze numerous Hutchinson Center research advances in cancer and other life-threatening diseases.

DESCRIPTION (provided by applicant): This application requests funds to purchase a Qiagen PyroMark Q96 MD Automated Pyrosequencing System to support over 10 NIH-funded projects involving researchers at the Fred Hutchinson Cancer Research Center (FHCRC). These projects address important areas of cancer research that include the identification and validation of genotypic and epigenetic markers that have the potential to predict the predisposition to various cancers and other genetic-based diseases. Pyrosequencing offers an accurate means to rapidly assess the proportion of mutated or epigenetically altered alleles in tissues in a high throughput manner. This information is critical in determining the biological and clinical relevance of these gene alterations in disease processes. Currently, there is no such instrument or similar capabilities for assessing both genetic alterations or polymorphisms and epigenetic alterations available to researchers in the Seattle area. If awarded, the PyroMark Q96 MD would be housed in the FHCRC Genomics Shared Resource, where expertise exists to operate, maintain, and manage the instrument, and a fee-based infrastructure is in place to ensure long-term support. The requested instrument will address both immediate and future needs, supporting translational NIH-funded research at the FHCRC.

DESCRIPTION (provided by applicant): Our genetic, molecular and biochemical analyses of ?-globin gene regulation have yielded novel and surprising results, including that the locus control region (LCR) is not required to initiate or propagate the open chromatin structure of the locus or confer stage-specific expression of the ?-like globin genes. In its native location, the primary role of the LCR is to enhance the transition from basal to activated transcription. We now propose to gain further insights into the molecular basis for initiating and propagating the chromatin and transcription states of the ?-globin locus during erythropoiesis. Our Specific Aims include: 1. Define epigenetic features including histone modifications, sequence co-localization, DNA methylation and candidate trans-acting factors involved in the initiation and propagation of active and silent states of the ?-globin locus. We will test the hypothesis that the ?-globin domain is larger and more dynamic than formerly appreciated, and that specific sequences, factors and epigenetic modifications are involved in the initiation and propagation of active and silent states of the locus. Initial studies, in collaboration with Dr. X-D Fu (UCSD) who developed the highly sensitive DNA Selection and Ligation (DSL) array-based method, will focus on a 1mb region of human chromosome 11 (h11) centered on the LCR, using a currently available array. We will design and build a similar array for analysis of the mouse locus. 2. Determine the role of cis- acting regulatory elements, transcription and cellular background on domains of histone modifications, factor binding and sequence co-localization in the ?-globin locus. We will use targeted mutation analyses in ES cell derived mice and our h11 transfer system to investigate several hypotheses regarding the regulation of p-globin gene expression. The effects of mutations on the epigenetic states of the locus, including factor binding, histone modifications and sequence interactions, will be determined by DSL analyses in stage-specific erythroid cells from mutant mice and in cell lines containing WT and modified h11s passed through different cellular backgrounds. Correlation of these results with activation state defined by primary transcript FISH, HS formation and the degree and extent of generalized DNase sensitivity will address several models of p-globin gene regulation. 3. Identify novel genes involved in erythroid maturation and p-globin gene expression. We will perform unbiased, genome-wide siRNA screens in the well- characterized G1E-ER cells and in ES derived erythroid progenitor (ES-EP) lines to identify novel factors involved in regulating p-globin gene expression and erythroid maturation. The screen makes use of a lentiviral siRNA library comprised of a redundant set of siRNAs for each known mouse gene. We propose several strategies for the validation, identification and prioritization of candidate siRNAs, as well as for the biochemical and functional analysis of genes identified.

Project Summary/Abstract. The proposal goal is to create a toolkit in which endogenous nuclear bodies (NBs) and chromatin compartments can be reversibly disrupted and endogenous genes can be moved among compartments. This toolkit will allow study of the functional interactions between NBs and chromatin and will be made widely available to the scientific community through the 4D Nucleome consortium. It is clear that chromatin reorganizes during differentiation, yet it is not well-understood how NBs interact with chromatin and influence this reorganization. For example, it has been difficult to study how organization of the repressive chromatin compartment might be seeded or affected by NBs such as the nucleolus because current tools are primarily limited to the study of exogenously expressed components and/or are not rapidly reversible. This toolkit will be developed in mouse embryonic stem cells (mESCs) because they are primary cells that can be differentiated into multiple cell types by end users. In Specific Aim 1, auxin inducible degrons (AIDs) will be employed to construct systems in which NBs and chromatin compartments are rapidly and reversibly disrupted. The AID sequence will be stably integrated into both alleles of endogenous genes coding for A) proteins essential for the integrity of the nucleolus (as a test NB) and B) lamina proteins that anchor peripheral heterochromatin (PH, as a test compartment). In contrast to current RNAi knockdown or cre/lox-based knockout techniques, this approach will allow rapid degradation of target proteins (minutes/hrs vs days), is not dependent on the cell cycle and is rapidly reversible. In Specific Aim 2, a novel modified TetO/TetR system will be used to tether specific proteins of interest to individual genes and move genes among compartments. Tethering of specific nucleolar and laminar structural proteins to both active and inactive mESC genes will be used as test cases and proteins that most effectively move genes among compartments will be identified. This tethering system will be more useful than current systems because it will not affect the chromatin context of the target region (as do large repetitive arrays) nor risk off-target effects (as in nuclease deficient dCas9-based approaches). Labeling unique genes is also simpler and quicker as compared to the transcription activator?like effector (TALE) approach, where TALEs targeting multiple sequences must be synthesized. In Specific Aim 3 we describe proof of principle experiments in differentiating cells to test the redundancy of the heterochromatic compartment and characterize microdomains within the PH. We demonstrate by a collaboration that other researchers are interested in these tools. Successful implementation of these tools by the general scientific community would permit detailed study of the role that NBs play in chromatin organization and provide tethering tools to study how genes move between nuclear compartments during both development and disease.

? DESCRIPTION (provided by applicant): Gene silencing is essential for normal development and protection of the genome from selfish genetic elements, such as retroposons. Perturbation of normal gene silencing results in genomic instability, impaired response to DNA damage, progression from normal to premalignant states, and eventually to cancer. While progress has been made in understanding silencing mechanisms in condensed heterochromatin, the silencing of genes in open euchromatin is less well understood. For example, recruitment of histone deacetylases (HDACs) to heterochromatic regions is essential for maintenance of the silent state. However, we have found that the mouse Mixed-Lineage Leukemia 5 (MLL5) protein and its Drosophila ortholog, UpSET, recruit HDACs to active genes to modulate open chromatin, fine-tune gene expression, and prevent inappropriate activation of silent neighboring genes. In addition, while histone turnover and the histone deposition machinery are essential for gene expression in differentiated cells, using novel methodology developed in our lab, we found that they are also key players in establishing gene silencing in embryonic stem cells (ESCs). Our proposed experiments use genetic, developmental, cell biology, biochemical, and molecular approaches in complementary systems (mice and flies) to determine the mechanisms underlying these novel regulatory pathways and elucidate their roles in hematopoiesis and stem cell biology. Specifically, we propose to: 1. Define the mechanisms by which MLL5/UpSET containing complexes modulate transcription. Using our novel protein tethering method we will determine the transcriptional role of mammalian MLL5 containing complexes in hematopoiesis. Taking advantage of the functional and structural conservation of the Drosophila ortholog of MLL5, UpSET, we will define how recruitment of these proteins modulates transcribed chromatin architecture around promoter regions to precisely orchestrate gene expression. 2. Elucidate the functional role of MLL5 in DNA damage response and repair. Like other proteins involved in gene silencing, MLL5 also participates in DNA damage repair. We will elucidate the role of MLL5 complexes in the DNA damage response and determine how mutation of Mll5 alters this response. We will also determine if lack of MLL5 increases tumor incidence or latency in tumor-sensitive backgrounds. Such results would provide the molecular basis for the DNA damage sensitivity of Mll5 null mice and association of poor prognosis preleukemic syndromes with haploinsufficiency of chromosome band 7q22 where human MLL5 resides. 3. Discover the molecular mechanisms by which SRCAP mediates silencing in ESCs and how perturbation of this silencing impacts hematopoiesis. We will elucidate the molecular basis of a previously unknown ESC silencing mechanism we recently discovered involving the Snf2-related CREBBP activator protein (SRCAP) H2A.Z deposition complex and polycomb repressive complex (PRC)1. We will determine how H2A.Z deposition by SRCAP underlies PRC1-mediated silencing and shapes the ESC and hematopoietic transcriptional landscapes.