Browsing by Subject "Chromatin"

Despite a largely static DNA sequence, our genomes are incredibly malleable. Comparative studies of chromatin features between different cell types, tissues, and species have revealed tremendous differences in how the genome is accessed, transcribed, and replicated. However, how the dynamics of chromatin accessibility contribute to development, environmental response, and disease status has only begun to be appreciated. In this work we identified chromatin accessibility changes by DNase-seq in three diverse processes: in granule neurons of the developing cerebellum, with intestinal epithelial cells in the absence of a normal microbiota, and with myelogenous leukemia cells in response to histone deacetylase inhibitor treatments. In all cases, we coupled these analyses with RNA-seq assays to identify concurrent transcriptional changes. By mapping the changes to these genome-wide signals we defined the contribution of local chromatin structure to the transcriptional programs underlying these processes, and improved our understanding of their relation to other chromatin changes like histone modifications. Furthermore we demonstrated use of the strongest accessibility changes to identify transcription factors critical for these processes by finding enrichment of their binding motifs. For a few of these key factors, depletion or overexpression of the protein was sufficient to regulate the expression of predicted target genes or exert limited chromatin accessibility changes, demonstrating the functional significance of these proteins in these processes. Together these studies have informed our understanding of the role chromatin accessibility changes play in development and environmental responses while also proving their utility for key regulator identification.

The accurate and timely replication of eukaryotic DNA during S-phase is of critical importance for the cell and for the inheritance of genetic information. Missteps in the replication program can activate cell cycle checkpoints or, worse, trigger the genomic instability and aneuploidy associated with diseases such as cancer. Eukaryotic DNA replication initiates asynchronously from hundreds to tens of thousands of replication origins spread across the genome. The origins are acted upon independently, but patterns emerge in the form of large-scale replication timing domains. Each of these origins must be localized, and the activation time determined by a system of signals that, though they have yet to be fully understood, are not dependent on the primary DNA sequence. This regulation of DNA replication has been shown to be extremely plastic, changing to fit the needs of cells in development or effected by replication stress.

We have investigated the role of chromatin in specifying the eukaryotic DNA replication program. Chromatin elements, including histone variants, histone modifications and nucleosome positioning, are an attractive candidate for DNA replication control, as they are not specified fully by sequence, and they can be modified to fit the unique needs of a cell without altering the DNA template. The origin recognition complex (ORC) specifies replication origin location by binding the DNA of origins. The S. cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. Next, we describe the D. melanogaster replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Taken together our data and analyses imply that the chromatin contains sufficient information to direct the DNA replication program.

DNA replication is an intricate process within eukaryotic cells that must be precisely executed to preserve genetic information. This process begins at multiple start sites, or origins of replication, along each chromosome which are selected, licensed, and activated through cell-cycle regulated steps. Powerful reconstitution studies have identified the proteins involved in these processes, but they do not fully recapitulate the nuclear environment. Within the nucleus, the genome is organized in a chromatin structure consisting of DNA and all associated factors. At origins of replication, local chromatin contributes to origin identity and activation, but the precise chromatin dynamics that occur at these sites during helicase activation and initial DNA unwinding have not been fully explored. Additionally, how these steps are regulated to ensure genomic stability remain unstudied within the context of chromatin.

To address these questions, I have developed a conditional system that removes polymerase α function to capture helicase activation at replication origins in the budding yeast. Under restrictive conditions, these cells (cdc17-ts-FRB) do not initiate replication. When allowed to recover, replication appears to initiate outside origins, necessitating a delay in G2/M phase to repair unreplicated gaps at origins. To investigate origin chromatin and helicase movement prior to replication, I used MNase chromatin profiling alongside ChIP-seq for various replication factors. Chromatin in a 1 kb region around early, efficient replication origins is disrupted under restrictive conditions. The active helicase unwinds DNA out to 1 kb from these origins and is likely the source of the chromatin disruption. I next used the cdc17-ts-FRB conditional system to investigate the regulation of helicase progression in the absence of replication. I first tested whether the intra-S-phase checkpoint had a role in stalling the helicase 1 kb from the origin. Though removing checkpoint activation distributed helicase movement and chromatin disruption to late, inefficient origins, it did not alter the distance the helicase progressed from the origin. Instead, the helicase stalls as it leaves the AT-rich origin region and encounters sequences with higher GC content. These results provide in vivo support for the recently proposed “dead man’s switch” model for decreased helicase processivity when uncoupled from replication.

Helicase activation and origin unwinding are essential steps during DNA replication that expose ssDNA and thus have the potential to cause genomic instability. My studies have captured origin chromatin dynamics caused by an active helicase unwinding DNA, and have contributed evidence that the helicase may be intrinsically less processive in the absence of leading strand synthesis. These results may have implications for the mechanisms underlying human diseases involving polymerase α, and contribute to our growing understanding of how the eukaryotic cell preserves the integrity of the genome.

Organisms are presented with a wide variety of environmental stimuli and must interpret and respond to these cues in to perform a wide variety of behaviors, such as foraging, mating, fleeing, and fighting. The ability of an organism to recognize various stimuli, such as pheromones, to identify mates or competitors through the activation of various circuits and molecular components in the brain is tightly regulated. In order to delineate how molecular changes occur in the brain during stimuli response we used Drosophila melanogaster as it has a well-defined nervous system. We focus in on the circuit which regulates sex-specific mating behaviors in male D. melanogaster. Sex-specific splicing regulates the expression of two genes known as fruitless (fruM) and doublesex (dsxM) in the courtship circuit. Here we demonstrate using in the fly olfactory system that Olfactory receptor 47b (Or47b) and Olfactory receptor 67d (Or67d) activity, through sensory experience, regulates the expression patterns of male-specific fruM through coincident activity of hormone binding transcription factors Gce and Met and histone acetyltransferase P300 activity. We also identify various genes which changes in various mutant and social contexts, including exon specific changes in fruitless transcripts as well as changes in the expression of hormone metabolism genes, and neuromodulators in antennae. Given these changes in neuromodulators and the known structure of the FruM and DsxM central circuits, we looked at changes in the chromatin state and expression levels and find changes in peripheral sensory neurons have downstream effects on higher order circuits. We identify that FruM regulates the chromatin structure of both itself and dsxM in whole brain lysates and that changes in chromatin structure depend on pheromone receptor and neurotransmitter activity across processing centers in the brain. Taken together, we identify potential candidates for future study, as well as lay the framework for understanding how sensory changes in the periphery have effects on various neuronal clusters in the brain.

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

The Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects more than >95% of the global adult population. In immunocompetent individuals, EBV infection is asymptomatic and takes place in the oral cavity, where EBV establishes a life-long latent infection in memory B cells by temporally regulating viral gene expression to mimic B cell maturation. In immunocompromised individuals, however, EBV infection can give rise to infectious mononucleosis, epithelial carcinomas, and lymphomas. To model EBV-mediated lymphomagenesis, infection of EBV in vitro generates growth-transformed and immortalized lymphoblastoid cell lines (LCLs), which allows for the characterization of dynamic viral and host gene expression. Our lab has found that the early phase after infection is transcriptionally distinct from the late phase when infected B cells are fully growth-transformed. We also found that apoptosis regulation in each phase of infection is uniquely regulated by a single viral nuclear protein that regulates host gene expression through epigenetic mechanisms. To determine if apoptosis regulation in EBV-infected B cells is virus-specific, I have characterized apoptosis regulation in uninfected maturing B cells and mitogen-stimulated B cells. For the upregulation of one anti-apoptotic protein, EBV infection promotes a chromatin structure resembling that in germinal center light zone B cells, indicating that EBV phenocopies germinal center chromatin regulation to promote apoptosis resistance. In addition to apoptosis regulation, EBV infection phenocopies various aspects of GC B cells and plasmablasts, where the inhibition of plasma cell differentiation increases the efficiency of immortalization and growth-transformation of B cells infected in vitro. The work outlined in this dissertation demonstrate that viral and host genes cooperate in mediating apoptosis regulation, differentiation, and ultimately fate-determination of EBV-infected B cells.

Heterochromatin, or condensed chromatin, is a transcriptionally repressive form of chromatin that occurs in many eukaryotic organisms. At its natural locations, heterochromatin is thought to play important roles in genome organization as well as gene expression. Just as important is the restriction of this repressive form of chromatin to appropriate regions of the genome. In the budding yeast Saccaromyces cerevisiae, domains of condensed, transcriptionally silenced chromatin are found at telomeres and at the silent-mating type cassettes, HML and HMR. At these locations, a complex of Silent Information Regulator (SIR) proteins gets recruited to DNA through discrete silencer elements. Once recruited, the Sir protein complex then spreads along chromosomes in a step-wise manner. This process results in the silencing of gene expression. It is unclear whether silenced chromatin is established in the same manner at different genomic locations. Understanding how silenced chromatin is formed is important for determining how these chromatin structures are regulated.

To better understand how silenced chromatin is established in different genomic contexts, I used chromatin immuoprecipitation to follow the rate of silenced chromatin formation at different locations. The rates of Sir protein assembly were compared at two locations, telomere VI-R and HMR. I discovered that the silencers at these two locations were equally proficient at recruiting Sir proteins. However, the rate of Sir protein assembly onto nucleosomes was far more rapid at HMR than at the telomere VI-R. Furthermore, the rate of Sir protein assembly was more rapid on one side of the HMR-E silencer at HMR than the other. Moreover, insertion of the HMR-E silencer adjacent to the telomere VI-R significantly improved the rate of Sir protein assembly onto nucleosomes. Additionally, observations that the association of Sir protein occurs simultaneously across several kilobases at HMR and that silencing at HMR is insensitive to co-expression of wild-type and catalytically inactive Sir2 proteins suggest that HMR-E enables the assembly of silenced chromatin in a non-linear fashion. These results suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases.

In addition to the HMR-E silencer, HMR is also characterized by the presence of a second auxiliary HMR-I silencer and a tRNA gene that functions as a boundary element to restrict the spread of silenced chromatin. I used chromatin immunoprecipitation to determine how each of these regulatory elements contribute to the steady-state levels of Sir protein association with chromatin. Consistent with a role for HMR-E beyond recruitment, I discovered that the HMR-E silencer alone promoted higher levels of Sir proteins on nucleosomes compared to the telomere VI-R. The levels of Sir protein association with HMR were further elevated by the HMR-I silencer, even though this silencer does not recruit Sir proteins on its own and does not contribute to any of the known functions of silenced chromatin at HMR. Additionally, although the tRNA gene did block the spread Sir proteins, I discovered that the capacity for Sir proteins to spread beyond a few kilobases was severely limited even in the absence of the boundary.

The results of this thesis work provide new insights into the mechanisms of silenced chromatin establishment and regulation in budding yeast. I show here that the capacity of Sir proteins to assemble onto nucleosomes is inherently limited. Additionally, silencers vary in their ability to promote this assembly. I conclude that the silencer is a key factor in determining the relative size, efficiency, and location of silenced chromatin domains in the cell.

In Saccharomyces cerevisiae, proper maintenance of haploid cell identity requires the SIR complex to mediate the silenced chromatin found at the cryptic mating-type loci, HML and HMR. This complex consists of Sir2, a histone deacetylase and the histone binding proteins Sir3 and Sir4. Interestingly, both Sir2 and Sir3 have paralogs from a genome duplication that occurred after the divergence of Saccharomyces and Kluyveromyces species. The histone deacetylase HST1 is the paralog of SIR2 and works with the promoter-specific SUM1 complex to repress sporulation and alpha-specific genes. ORC1 is the paralog of SIR3 and is an essential subunit of the Origin Recognition Complex and also recruits SIR proteins to the HM loci. I have investigated the functions of these proteins in the non-duplicated species Kluyveromyces lactis and compared these functions to those found in S. cerevisiae.

I have shown that SIR2 and HST1 subfunctionalized post-duplication via the duplication, degeneration and complementation mechanism. In S. cerevisiae, Sir2 has retained the ability to function like Hst1 when in an hst1Δ strain. I have also shown, with a chimeric Sir2-Hst1 protein, that there are distinct specificity domains for Sir2 interaction with the SIR complex and Hst1 interaction with the SUM1 complex that have diverged between Sir2 and Hst1. Trans-species complementation assays show that the non-duplicated Sir2 from K. lactis can interact with both SIR and SUM1 complexes in S. cerevisiae.

Further analysis into the non-duplicated experimental system of K. lactis has revealed that deletion of KlSir2 de-represses the HM loci as well as sporulation and cell-type specific genes. A physical interaction between KlSir2 and the histone binding protein KlSir4 is conserved in K. lactis, and both proteins spread across the HML locus and associate with telomeres in a manner similar to S. cerevisiae. KlSir2 also physically interacts with the DNA-binding protein, KlSum1, to repress sporulation and cell-type specific genes in a promoter-specific manner and recruitment of KlSir2 to these loci is dependent on KlSum1. Surprisingly, deletion of KlSUM1 also de-represses HML and HMR, a phenotype not observed in S. cerevisiae. I show by chromatin immunoprecipitation that KlSum1 directly regulates the HM loci by spreading across these regions in a mechanism that is distinct from its role in repressing sporulation-specific genes. This result indicates that KlSum1 is a key regulator of not only meiotic, but also mating-type transcriptional programming.

The SIR3-ORC1 gene pair has previously been used as an example of neofunctionalization based on accelerated rates of evolution. However, my studies of KlOrc1 show it is distributed across HML and associates with Sir2 and Sir4 at telomeres, indicative of it having Sir3-like capabilities to spread across chromatin. This ability of KlOrc1 to spread is distinct from its functions with ORC, and is entirely dependent on its BAH domain. These findings demonstrate that prior to the genome duplication there was a silencing complex that contained both KlSir2 and KlOrc1. In addition to their functions at HML and the telomeres, KlOrc1 associates with replication origins and KlSir2 and KlSum1 work in complex to repress sporulation genes in a promoter-specific manner. The multiple functions of both KlOrc1 and KlSir2 in K. lactis indicate that after duplication, these properties were divided among paralogs and subsequently specialized to perform the functions that have been characterized in S. cerevisiae.

All DNA-templated events, including replication and gene transcription, occur in the context of the local chromatin environment. The passage of the replication machinery results in disassembly of chromatin, which must be re-assembled behind the replication fork to re-establish the epigenetic state of the cell. Many of the factors and mechanisms regulating DNA replication and chromatin assembly have been identified from elegant in vitro biochemical experiments, work in model systems like Saccharomyces cerevisiae, or novel proteomic approaches. In spite of current advances in the field, it is still not clear how the chromatin landscape is organized and re-assembled during this process.

Current methods, while informative, lack the genome-wide base-pair resolution required to assess the dynamics of chromatin assembly and maturation in a spatial-temporal manner. To overcome the limitations of these studies, I have taken advantage of an epigenome mapping technique based on micrococcal nuclease (MNase) digestion followed by paired-end sequencing. This approach facilitates the analysis of chromatin structure by capturing not only nucleosomes, but also smaller DNA binding protein footprints in a factor-agnostic manner. I have developed a technique based on this approach that generates Nascent Chromatin Occupancy Profiles (NCOPs) to study the dynamics of chromatin assembly following passage of the DNA replication fork at a genome-wide level and at single base-pair resolution in S. cerevisiae. It employs a nucleoside analog to specifically enrich for nascent chromatin, which can be captured following a chase over different periods of time. Thus, NCOPs resolve the structure of nascent and mature chromatin, facilitating the analysis of chromatin maturation across the entire genome.

Using NCOPs, I provide a comprehensive description of the maturation process across different genomic regions and the dynamics of small DNA binding factor association with nascent and mature chromatin states. Our results support previous work characterizing the structure of nascent chromatin as being more disorganized and having poorly positioned nucleosomes. Importantly, using positioning and occupancy scores, I provide new details on the structure of nascent and mature chromatin at intergenic regions, including replication origins, and at highly transcribed and poorly transcribed genes. I uncovered that local epigenetic footprints have the potential to shape the dynamics of chromatin assembly, generating a chromatin maturation landscape that is dependent on the parental chromatin. Finally, I resolved patterns of transcription factor occupancy with nascent and mature chromatin, and observed transient factor association in the nascent state.

In all, this work provides insight into the dynamics of chromatin assembly, and allows for genome-wide and base-pair resolution investigation of chromatin maturation. The genomic and bioinformatic approaches developed here open the door for further investigation of the dynamics of epigenetic inheritance and the role of known and unknown players in re-establishing the eukaryotic epigenome following passage of the DNA replication fork.

Eukaryotic genomes have extensive flexibility and plasticity to modify transcription and replication programs, yielding a myriad of differentiated cell types and survival mechanisms to adverse environmental conditions. As these genomic processes require precise localization of DNA-binding factors, their dynamic temporal and spatial distributions provide dramatically different interpretations of a static genome sequence. DNA-binding factors must compete with nucleosomes, the basic subunit of chromatin, for access to the underlying DNA sequence. Even though the spatial preferences of these proteins are partially explained by DNA sequence alone, the complete genome occupancy profile has remained elusive, and we currently have a limited understanding of how DNA-binding protein configurations directly impact transcription and replication function.

Profiling the entire chromatin environment has typically required multiple experiments to capture both DNA-binding factors and nucleosomes. Here, we have extended the traditional micrococcal nuclease (MNase) digestion assay to simultaneously resolve both nucleosomes and smaller DNA-binding footprints in Saccharomyces cerevisiae. Visualization of protected DNA fragments revealed a nucleotide-resolution view of the chromatin architecture at individual genomic loci. We show that different MNase digestion times can capture nucleosomes partially unwrapped or complexed with chromatin remodelers. Stereotypical DNA-binding footprints are evident across all promoters, even at low-transcribed and silent genes. By aggregating the chromatin profiles across transcription-factor--binding sites, we precisely resolve protein footprints, yielding in vivo insights into protein-DNA interactions. Together, our MNase method, in one experiment, provides an unprecedented assessment of the entire chromatin structure genome-wide.

We utilized this approach to interrogate how the replication program is regulated by the chromatin environment surrounding DNA replication initiation sites. Pre-replicative complex (pre-RC) formation commences with recruitment of the origin recognition complex (ORC) to specific locations in the genome, termed replication origins. Although successful pre-RC assembly primes each site for S-phase initiation by loading the Mcm2-7 helicase, replication origins have substantially different activation times and efficiencies. We posited that replication origin function is substantially impacted by the local chromatin environment. Here, we resolved a high-resolution ORC-dependent footprint at 269 replication origins genome-wide. Even though ORC in S. cerevisiae remains bound at replication origins throughout the cell cycle, we detected a subset of inefficient origins that did not yield a footprint until G1, suggesting a more transient ORC interaction prior to pre-RC assembly. Nucleosome movement accommodated the pre-RC-induced expansion of the ORC-dependent footprint in G1, leading to increased activation efficiency. Mcm2-7 loading is preferentially directed to one side of each replication origin, in close proximity to the origin-flanking nucleosome. Our data demonstrates that pre-RC components are assembled into multiple configurations in vivo.

We anticipate that extending chromatin occupancy profiling to many different cell types will reveal further insights into genome regulation.