Chromosome Binding Research Articles

RNA-Seq analysis and whole genome DNA-binding profile of the Vibrio cholerae histone-like nucleoid structuring protein (H-NS).

The data described in this article pertain to the genome-wide transcription profiling of a Vibrio cholerae mutant lacking the histone-like nucleoid structuring protein (H-NS) and the mapping of the H-NS chromosome binding sites (Wang et al., 2015 [1]; Ayala et al., 2015 [2]). H-NS is a nucleoid-associated protein with two interrelated functions: organization of the bacterial nucleoid and transcriptional silencing (Dorman, 2013 [3]). Both functions require DNA binding and protein oligomerization (Spurio et al., 1997[4]; Dame et al., 2001 [5]). H-NS commonly silences the expression of virulence factors acquired by lateral gene transfer (Navarre et al., 2006 [6]). The highly pleiotropic nature of hns mutants in V. cholerae indicates that H-NS impacts a broad range of cellular processes such as virulence, stress response, surface attachment, biofilm development, motility and chemotaxis. We used a V. cholerae strain harboring a deletion of hns and a strain expressing H-NS tagged at the C-terminus with the FLAG epitope to generate datasets representing the hns transcriptome and DNA binding profile under laboratory conditions (LB medium, 37 °C). The datasets are publicly available at the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE62785 and GSE64249.

Research progress of high mobility group protein B1 in hematologic malignancies

High mobility group protein B1 (HMGB1) belongs to non-histone chromosomal binding protein that exists in eukaryotic nuclei. Early studies have shown that HMGB1 acted as a late inflammatory mediator involved in inflammation progress. In recent years, it is found highly expressed in many hematologic malignancies, and it participates in the development and drug resistance of hematologic malignancies through a variety of ways. This article reviews the relationship between HMGB1 and tumor multidrug resistance, leukemia, lymphoma and multiple myeloma, in order to provide a new target for clinical treatment of hematologic malignancies. Key words: HMGB1 protein; Leukemia; Lymphoma; Multiple myeloma

Phosphorylation of HPV-16 E2 at serine 243 enables binding to Brd4 and mitotic chromosomes.

The papillomavirus E2 protein is involved in the maintenance of persistent infection and known to bind either to cellular factors or directly to mitotic chromosomes in order to partition the viral genome into the daughter cells. However, how the HPV-16 E2 protein acts to facilitate partitioning of the viral genome remains unclear. In this study, we found that serine 243 of HPV-16 E2, located in the hinge region, is crucial for chromosome binding during mitosis. Bromodomain protein 4 (Brd4) has been identified as a cellular binding target through which the E2 protein of bovine papillomavirus type 1 (BPV-1) tethers the viral genome to mitotic chromosomes. Mutation analysis showed that, when the residue serine 243 was substituted by glutamic acid or aspartic acid, whose negative charges mimic the effect of constitutive phosphorylation, the protein still can interact with Brd4 and colocalize with Brd4 in condensed metaphase and anaphase chromosomes. However, substitution by the polar uncharged residues asparagine or glutamine abrogated Brd4 and mitotic chromosome binding. Moreover, following treatment with the inhibitor JQ1 to release Brd4 from the chromosomes, Brd4 and E2 formed punctate foci separate from the chromosomes, further supporting the hypothesis that the association of the HPV-16 E2 protein with the chromosomes is Brd4-dependent. In addition, the S243A E2 protein has a shorter half-life than the wild type, indicating that phosphorylation of the HPV-16 E2 protein at serine 243 also increases its half-life. Thus, phosphorylation of serine 243 in the hinge region of HPV-16 E2 is essential for interaction with Brd4 and required for host chromosome binding.

Abstract 5083: Metaphase delays lead to chromosome cohesion fatigue, a novel mechanism underlying chromosome instability and aneuploidy

Abstract Cohesin fatigue describes our findings that cells delayed at metaphase undergo asynchronous separation of chromatids. We found that these unpaired chromatids randomly migrate on spindle microtubules or become merotelic, wherein a single centromere attaches to spindle fibers from both poles. Many cells eventually exit mitosis after cohesion fatigue, and the unpaired chromatids can form micronuclei, can rupture, and can mis-segregate. These results suggest that cohesion fatigue may cause gain or loss of whole chromosomes (numerical aneuploidy) as well as duplications, deletions, or translocations of large chromosome fragments (segmental aneuploidy). Numerical and segmental aneuploidies are hallmarks of cancer cells, often occurring together, and cohesion fatigue is a potential mechanism for simultaneously generating both. Any induced metaphase delay will lead to cohesion fatigue as long as spindle function is intact. Among mitotic defects, cohesion fatigue may be relatively common, but has often gone unrecognized since its detection requires continuous tracking of chromosome behavior. We have observed that cohesion fatigue is induced after metaphase arrest caused by RNAi-mediated depletion of the Ska (Spindle and kinetochore-associated) complex. Recently we discovered the underlying mechanism for the metaphase delay in Ska-depleted cells. We find that the Ska complex promotes chromosome binding and activity of the Anaphase-Promoting Complex/Cyclosome, the E3 ubiquitin ligase that controls anaphase onset and mitotic exit. Cohesion fatigue is also induced by certain drugs such as proteasome inhibitors, which are under development for cancer therapy. Using proteasome inhibition we found that cohesion fatigue initiates at centromeres and progresses along chromosome arms. While complete chromatid separation may occur after hours of metaphase arrest, our recent tracking reveals that abnormal centromere separation occurs after a few minutes of arrest. This early loss of centromere cohesion may lead to mitotic defects in cells that experience even a brief metaphase delay. Cohesion fatigue is not observed in cells arrested in mitosis with microtubule poisons because outward pulling forces on chromosomes are lost. In normal dividing cells, spontaneous mitotic defects are relatively rare, but these are substantially increased in transformed cells. Genes coding for components and regulators of the cohesin complex, which mediates sister chromatid cohesion, are among the most highly mutated genes in tumors. We propose that cohesin defects in cancer cells may exacerbate cohesion fatigue leading to increased chromosome instability. In carcinogenesis, cells that undergo metaphase delays are likely to generate mitotic errors through cohesion fatigue that result in numerical and segmental aneuploidy. Citation Format: John R. Daum, Sushama Sivakumar, Aaron R. Tipton, Michael W. Davidson, Gary J. Gorbsky. Metaphase delays lead to chromosome cohesion fatigue, a novel mechanism underlying chromosome instability and aneuploidy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5083. doi:10.1158/1538-7445.AM2014-5083

The maintenance of chromosome structure: positioning and functioning of SMC complexes.

Structural maintenance of chromosomes (SMC) complexes, which in eukaryotic cells include cohesin, condensin and the Smc5/6 complex, are central regulators of chromosome dynamics and control sister chromatid cohesion, chromosome condensation, DNA replication, DNA repair and transcription. Even though the molecular mechanisms that lead to this large range of functions are still unclear, it has been established that the complexes execute their functions through their association with chromosomal DNA. A large set of data also indicates that SMC complexes work as intermolecular and intramolecular linkers of DNA. When combining these insights with results from ongoing analyses of their chromosomal binding, and how this interaction influences the structure and dynamics of chromosomes, a picture of how SMC complexes carry out their many functions starts to emerge.

A transient α-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1

The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays show that the defects of the proline mutants are the result of impaired binding of Top3 and Rmi1 to Sgs1. Extending mutagenesis N-terminally revealed a second functionally critical region that spans residues 9–17. Depending on the position of the proline substitution in the helix functional impairment of Sgs1 function varied, gradually increasing from the C- to the N-terminus. The multiscale approach we used to interrogate structure/function relationships in the long disordered N-terminal segment of Sgs1 allowed us to precisely define a functionally critical region and should be generally applicable to other disordered proteins.

Interaction between Basic Residues of Epstein-Barr Virus EBNA1 Protein and Cellular Chromatin Mediates Viral Plasmid Maintenance

The Epstein-Barr virus (EBV) genome is episomally maintained in latently infected cells. The viral protein EBNA1 is a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes (chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanism by which the CBDs of EBNA1 attach to cellular chromatin is still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 (amino acids 40-54) and CBD2 (amino acids 328-377) regions with alanines progressively impaired chromosome binding activity of EBNA1. The complete arginine-to-alanine substitutions within the CBD1 and -2 regions abolished the ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effect, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine-rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, whereas a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between the basic amino acids within the CBDs and negatively charged cellular chromatin.

Transcription factor binding kinetics constrain noise suppression via negative feedback

Negative autoregulation, where a transcription factor regulates its own expression by preventing transcription, is commonly used to suppress fluctuations in gene expression. Recent single molecule in vivo imaging has shown that it takes significant time for a transcription factor molecule to bind its chromosomal binding site. Given the slow association kinetics, transcription factor mediated feedback cannot at the same time be fast and strong. Here we show that with a limited association rate follows an optimal transcription factor binding strength where noise is maximally suppressed. At the optimal binding strength the binding site is free a fixed fraction of the time independent of the transcription factor concentration. One consequence is that high-copy number transcription factors should bind weakly to their operators, which is observed for transcription factors in Escherichia coli. The results demonstrate that a binding site's strength may be uncorrelated to its functional importance.

Functions of the Hha and YdgT Proteins in Transcriptional Silencing by the Nucleoid Proteins, H-NS and StpA, in Escherichia coli

The Hha and YdgT proteins are suggested to modulate the expression of horizontally acquired genes by interacting with H-NS and StpA, which play central roles in the transcriptional silencing of such genes. However, it is also possible that Hha/YdgT repress gene expression independently of H-NS/StpA, as we have not fully understood the molecular mechanism through which Hha/YdgT modulate H-NS/StpA activity. To gain further insight into the basic functions of Hha/YdgT, we analysed the impact of hha/ydgT double inactivation on the transcriptome profile of Escherichia coli K-12, and compared the effects with that of hns/stpA double inactivation. In addition, we examined the effects of hha/ydgT inactivation on the chromosomal binding of H-NS, and conversely the effects of hns/stpA inactivation on the chromosomal binding of Hha. Our results demonstrated that the chromosomal binding of Hha requires H-NS/StpA, and is necessary for the repression of a subset of genes in the H-NS/StpA regulon. Furthermore, the distribution of H-NS binding around Hha/YdgT-dependent and -independent genes suggests that Hha/YdgT proteins modulate formation of the H-NS/StpA-DNA complex.

Activating the genome during development and exit from mitosis

During mitosis, the genome is silent. When cells exit mitosis and re-enter the cell cycle, how does the transcriptional program of a differentiated cell become reactivated faithfully and in a timely manner? We previously found that in undifferentiated progenitor cells in the early embryo, the competence to activate transcriptionally silent genes is established by pioneer transcription factors, such as FoxA proteins. Pioneer factors have the ability to bind their target DNA sequence in compacted chromatin and allow other factors to bind nearby. We discovered that the same pioneer transcription factors that function in early liver development remain bound to the genome during liver cell mitosis, when the genome compacts, most factors and RNA polymerase are excluded, and transcription is silent. Specifically, the pioneer transcription factor FoxA1 exhibits virtually complete mitotic chromosome binding, whereas other liver factors bind with a range of efficiencies and binding modalities. The evident hierarchy of specific binding, nonspecific binding, partial chromatin binding, failure to bind mitotic chromosomes, and mitotic instability reflects the factors’ developmental roles in gene activation, and suggest that re-activation of the genome following mitosis involves a replay of the network by which the cell type was established in development.

Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes

While most transcription factors exit the chromatin during mitosis and the genome becomes silent, a subset of factors remains and “bookmark” genes for rapid re-activation as cells progress through the cell cycle. Yet it is unknown whether such “bookmarking” factors bind to chromatin similarly in mitosis and how different binding capacities among them relate to function. We compared a diverse set of transcription factors involved in liver differentiation and found markedly different extents of mitotic chromosome binding. Among them, only the pioneer factor FoxA1 exhibits virtually complete mitotic chromosome binding. Genomically, about 15% of the FoxA1 interphase target sites are bound in mitosis, including at genes for liver differentiation. Biophysical, genome mapping, and mutagenesis studies of FoxA1 reveals two different modes of binding to mitotic chromatin: Specific binding, at sites that continue to be bound from interphase, and nonspecific binding, whereby due to intrinsic chromatin affinity, FoxA1 remains in the genomic vicinity of its interphase target genes. Both specific and nonspecific binding contribute to timely reactivation of target genes, post mitosis. These studies reveal an unexpected diversity in the mechanisms by which transcription factors help retain cell identity during mitosis.

Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes

While most transcription factors exit the chromatin during mitosis and the genome becomes silent, a subset of factors remains and "bookmarks" genes for rapid reactivation as cells progress through the cell cycle. However, it is unknown whether such bookmarking factors bind to chromatin similarly in mitosis and how different binding capacities among them relate to function. We compared a diverse set of transcription factors involved in liver differentiation and found markedly different extents of mitotic chromosome binding. Among them, the pioneer factor FoxA1 exhibits the greatest extent of mitotic chromosome binding. Genomically, ~15% of the FoxA1 interphase target sites are bound in mitosis, including at genes that are important for liver differentiation. Biophysical, genome mapping, and mutagenesis studies of FoxA1 reveals two different modes of binding to mitotic chromatin. Specific binding in mitosis occurs at sites that continue to be bound from interphase. Nonspecific binding in mitosis occurs across the chromosome due to the intrinsic chromatin affinity of FoxA1. Both specific and nonspecific binding contribute to timely reactivation of target genes post-mitosis. These studies reveal an unexpected diversity in the mechanisms by which transcription factors help retain cell identity during mitosis.

Different chromatin interfaces of the Drosophila dosage compensation complex revealed by high-shear ChIP-seq

Transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila males is brought about by a ribonucleoprotein assembly called Male-Specific-Lethal or Dosage Compensation Complex (MSL-DCC). This machinery is formed in male flies and specifically associates with active genes on the X chromosome. After assembly at dedicated high-affinity “entry” sites (HAS) on the X chromosome, the complex distributes to the nearby active chromatin. High-resolution, genome-wide mapping of the MSL-DCC subunits by chromatin immunoprecipitation (ChIP) on oligonucleotide tiling arrays suggests a rather homogenous spreading of the intact complex onto transcribed chromatin. Coupling ChIP to deep sequencing (ChIP-seq) promises to map the chromosomal interactions of the DCC with improved resolution. We present ChIP-seq binding profiles for all complex subunits, including the first description of the RNA helicase MLE binding pattern. Exploiting the preferential representation of direct chromatin contacts upon high-energy shearing, we report a surprising functional and topological separation of MSL protein contacts at three classes of chromosomal binding sites. Furthermore, precise determination of DNA fragment lengths by paired-end ChIP-seq allows decrypting of the local complex architecture. Primary contacts of MSL-2 and MLE define HAS for the DCC. In contrast, association of the DCC with actively transcribed gene bodies is mediated by MSL-3 binding to nucleosomes. We identify robust MSL-1/MOF binding at a fraction of active promoters genome-wide. Correlation analyses suggest that this association reflects a function outside dosage compensation. Our comprehensive analysis provides a new level of information on different interaction modes of a multiprotein complex at distinct regions within the genome.

Biochemical Characterization of Diverse Stat5b-Binding Enhancers That Mediate Growth Hormone-Activated Insulin-Like Growth Factor-I Gene Transcription

Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH.

MiR290-5p/292-5p Activate the Immunoglobulin kappa Locus in B Cell Development

Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.

Phosphorylation Regulates Binding of the Human Papillomavirus Type 8 E2 Protein to Host Chromosomes

The papillomavirus E2 proteins are indispensable for the viral life cycle, and their functions are subject to tight regulation. The E2 proteins undergo posttranslational modifications that regulate their properties and roles in viral transcription, replication, and genome maintenance. During persistent infection, the E2 proteins from many papillomaviruses act as molecular bridges that tether the viral genomes to host chromosomes to retain them within the host nucleus and to partition them to daughter cells. The betapapillomavirus E2 proteins bind to pericentromeric regions of host mitotic chromosomes, including the ribosomal DNA loci. We recently reported that two residues (arginine 250 and serine 253) within the chromosome binding region of the human papillomavirus type 8 (HPV8) E2 protein are required for this binding. In this study, we show that serine 253 is phosphorylated, most likely by protein kinase A, and this modulates the interaction of the E2 protein with cellular chromatin. Furthermore, we show that this phosphorylation occurs in S phase, increases the half-life of the E2 protein, and promotes chromatin binding from S phase through mitosis.

Cell Size Control in Bacteria

Like eukaryotes, bacteria must coordinate division with growth to ensure cells are the appropriate size for a given environmental condition or developmental fate. As single-celled organisms, nutrient availability is one of the strongest influences on bacterial cell size. Classic physiological experiments conducted over four decades ago first demonstrated that cell size is directly correlated with nutrient source and growth rate in the Gram-negative bacterium Salmonella typhimurium. This observation subsequently served as the basis for studies revealing a role for cell size in cell cycle progression in a closely related organism, Escherichia coli. More recently, the development of powerful genetic, molecular, and imaging tools has allowed us to identify and characterize the nutrient-dependent pathway responsible for coordinating cell division and cell size with growth rate in the Gram-positive model organism Bacillus subtilis. Here, we discuss the role of cell size in bacterial growth and development and propose a broadly applicable model for cell size control in this important and highly divergent domain of life.

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation. The N-terminal domain of a cytoplasmic chemoreceptor encoded next to ppfA is also required for segregation, probably functioning as a ParB analog to control PpfA ATPase activity. An orphan ParA involved in segregating protein clusters therefore uses a similar mechanism to plasmid-segregating ParA/B systems and requires a partner protein for function. Given the large number of genomes that encode orphan ParAs, this may be a common mechanism regulating segregation of proteins and protein complexes.

Activating the genome during development and exit from mitosis

We are interested in the means by which transcription factors, inductive signals, and chromatin states control the specification of different mammalian cell types. We found that pioneer transcription factors endow the competence upon multipotent progenitor cells to activate particular cell‐specific genetic programs in development. Recently, we discovered that the same pioneer transcription factors that function in development remain bound to the genome during mitosis, when most factors and RNA polymerase are excluded and transcription is silent. Transcriptional re‐activation of the genome, following mitosis, requires re‐setting of cell‐specific programs that were initially established during development. We compared a diverse set of transcription factors involved in liver differentiation and find different modes of mitotic chromosome binding. The pioneer transcription factor FoxA1 exhibits virtually complete mitotic chromosome binding, whereas other liver factors bind with a range of efficiencies and binding modalities. The evident hierarchy of specific binding, storage site binding, partial chromatin binding, failure to bind mitotic chromosomes, and mitotic instability reflects the factors’ developmental roles in gene activation, and suggest that re‐activation of the genome following mitosis involves a replay of the network by which the cell type was established in development.

CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.