DNA in Cell

1 . Epigenome Sequencing Comes of Age

Jian-Kang Zhu Cell 133: 395-397.[Full Text] [PDF]

Epigenetic states are responsive to developmental and environmental signals, and as a consequence a eukaryotic cell can have many different epigenomes. In this issue of Cell, Lister et al., 2008Chan et al., 2006Collart et al., Danilova et al., 2007Nudler et al., 2002Bernstein et al., 2007Gnatt et al., 2001Lee et al., 2005Bicknell et al., 2004Brenner, 1974Bishop et al., 1992Bernstein et al., 2005Blat et al., 1999Dyda et al., 1994Cheung et al., 2000Burley et al., 1985Chen et al., 2005Benard et al., 2001Ebersole et al., 2000 present the floral epigenome of Arabidopsis using next-generation sequencing technology to analyze both DNA methylation at single-base resolution and the expression of small RNAs.

2 . Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis

Ryan Lister, Ronan C. O'Malley, Julian Tonti-Filippini, Brian D. Gregory, Charles C. Berry, A. Harvey Millar and Joseph R. Ecker Cell : .[Full Text] [PDF]

Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

3 . MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks

Manuel Stucki, Julie A. Clapperton, Duaa Mohammad, Michael B. Yaffe, Stephen J. Smerdon and Stephen P. Jackson Cell 133: 549.[Full Text] [PDF]

4 . Meiosis I Is Established through Division-Specific Translational Control of a Cyclin

Thomas M. Carlile and Angelika Amon Cell 133: 280-291.[Full Text] [PDF]

In budding yeast, key meiotic events such as DNA replication, recombination, and the meiotic divisions are controlled by Clb cyclin-dependent kinases (Clb-CDKs). Using a novel synchronization procedure, we have characterized the activity of these Clb-CDKs and observed a surprising diversity in their regulation during the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II, through 5′UTR-mediated translational control of its transcript. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore defines Clb3 as an inhibitor of the meiosis I chromosome segregation program. Our results demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.

5 . Independent Positioning and Action of Escherichia coli Replisomes in Live Cells

Rodrigo Reyes-Lamothe, Christophe Possoz, Olessia Danilova and David J. Sherratt Cell 133: 90-102.[Full Text] [PDF]

A prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.

6 . Transcription Termination: Pulling Out All the Stops

Jack F. Greenblatt Cell 132: 917-918.[Full Text] [PDF]

In this issue, Huettel et al., 2006Cao et al., 2002Cohen-Fix et al., 1996Cook, 1999Larson et al., 2008Bernstein et al., 2006Davenport et al., 2000Hunter, Berger et al., Booth et al., 2001Bailis et al., 1998Bernstein et al., 2004Birney et al., 2007DeLano, 2002Caretti et al., 2003Bunting et al., 2003Chartier-Harlin et al., 2004Bakkenist et al., 2004Earnshaw et al., 1989 describe the use of optical traps to pull on the DNA template or RNA transcript and thereby explore the termination mechanism for E. coli RNA polymerase at intrinsic terminators. Their results imply that, depending on the nature of the terminator sequence, RNA polymerase uses either hypertranslocation or RNA:DNA shearing to destabilize the hybrid in the transcription bubble.

7 . FoxA1 Translates Epigenetic Signatures into Enhancer-Driven Lineage-Specific Transcription

Mathieu Lupien, Jérôme Eeckhoute, Clifford A. Meyer, Qianben Wang, Yong Zhang, Wei Li, Jason S. Carroll, X. Shirley Liu and Myles Brown Cell 132: 958-970.[Full Text] [PDF]

Complex organisms require tissue-specific transcriptional programs, yet little is known about how these are established. The transcription factor FoxA1 is thought to contribute to gene regulation through its ability to act as a pioneer factor binding to nucleosomal DNA. Through genome-wide positional analyses, we demonstrate that FoxA1 cell type-specific functions rely primarily on differential recruitment to chromatin predominantly at distant enhancers rather than proximal promoters. This differential recruitment leads to cell type-specific changes in chromatin structure and functional collaboration with lineage-specific transcription factors. Despite the ability of FoxA1 to bind nucleosomes, its differential binding to chromatin sites is dependent on the distribution of histone H3 lysine 4 dimethylation. Together, our results suggest that methylation of histone H3 lysine 4 is part of the epigenetic signature that defines lineage-specific FoxA1 recruitment sites in chromatin. FoxA1 translates this epigenetic signature into changes in chromatin structure thereby establishing lineage-specific transcriptional enhancers and programs.

8 . Applied Force Reveals Mechanistic and Energetic Details of Transcription Termination

Matthew H. Larson, William J. Greenleaf, Robert Landick and Steven M. Block Cell 132: 971-982.[Full Text] [PDF]

Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2–3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.

9 . Hop1 and the Meiotic DNA-Damage Response

Neil Hunter Cell 132: 731-732.[Full Text] [PDF]

During the DNA-damage response, adaptor proteins mediate signaling between the PI3K-like sensor kinases, ATM and ATR, and serine/threonine effector kinases. Cokus et al., 2008Bernstein et al., 2007Bloom et al., 2007Bates et al., 2005Gusarov et al., 1999Badve et al., 2007Adelman et al., 2002Carballo et al., 2008Agius et al., 2006Anderson et al., 2002Anuradha et al., 2004Albert et al., 2007Ansel et al., 2003Brünger et al., 1998Brown et al., 2003Argiriadi et al., 2006Blanco-Rodríguez, 2002Ahmed et al., 2001Chan et al., 2005 now show that the chromosomal protein Hop1 mediates PI3K-like kinase signaling during the repair of DNA double-strand breaks (DSBs) in meiosis.

10 . Cellular Programming of Plant Gene Imprinting

Jin Hoe Huh, Matthew J. Bauer, Tzung-Fu Hsieh and Robert L. Fischer Cell 132: 735-744.[Full Text] [PDF]

Gene imprinting, the differential expression of maternal and paternal alleles, independently evolved in mammals and in flowering plants. A unique feature of flowering plants is a double-fertilization event in which the sperm fertilize not only the egg, which forms the embryo, but also the central cell, which develops into the endosperm (an embryo-supporting tissue). The distinctive mechanisms of gene imprinting in the endosperm, which involve DNA demethylation and histone methylation, begin in the central cell and sperm prior to fertilization. Flowering plants might have coevolved double fertilization and imprinting to prevent parthenogenetic development of the endosperm.

11 . C. elegans Telomeres Contain G-Strand and C-Strand Overhangs that Are Bound by Distinct Proteins

Marcela Raices, Ramiro E. Verdun, Sarah A. Compton, Candy I. Haggblom, Jack D. Griffith, Andrew Dillin and Jan Karlseder Cell 132: 745-757.[Full Text] [PDF]

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3′ G-strand overhangs as well as 5′ C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5′ and 3′ single-stranded termini.

12 . Phosphorylation of the Axial Element Protein Hop1 by Mec1/Tel1 Ensures Meiotic Interhomolog Recombination

Jesús A. Carballo, Anthony L. Johnson, Steven G. Sedgwick and Rita S. Cha Cell 132: 758-770.[Full Text] [PDF]

An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.

13 . Dynamic Regulation of Nucleosome Positioning in the Human Genome

Dustin E. Schones, Kairong Cui, Suresh Cuddapah, Tae-Young Roh, Artem Barski, Zhibin Wang, Gang Wei and Keji Zhao Cell 132: 887-898.[Full Text] [PDF]

The positioning of nucleosomes with respect to DNA plays an important role in regulating transcription. However, nucleosome mapping has been performed for only limited genomic regions in humans. We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Solexa high-throughput sequencing technique. We find that nucleosome phasing relative to the transcription start sites is directly correlated to RNA polymerase II (Pol II) binding. Furthermore, the first nucleosome downstream of a start site exhibits differential positioning in active and silent genes. TCR signaling induces extensive nucleosome reorganization in promoters and enhancers to allow transcriptional activation or repression. Our results suggest that H2A.Z-containing and modified nucleosomes are preferentially lost from the −1 nucleosome position. Our data provide a comprehensive view of the nucleosome landscape and its dynamic regulation in the human genome.

14 . Cohesins Functionally Associate with CTCF on Mammalian Chromosome Arms

Vania Parelho, Suzana Hadjur, Mikhail Spivakov, Marion Leleu, Stephan Sauer, Heather C. Gregson, Adam Jarmuz, Claudia Canzonetta, Zoe Webster, Tatyana Nesterova, Bradley S. Cobb, Kyoko Yokomori, Niall Dillon, Luis Aragon, Amanda G. Fisher and Matthias Merkenschlager Cell 132: 422-433.[Full Text] [PDF]

Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.

15 . Mechanism of IS200/IS605 Family DNA Transposases: Activation and Transposon-Directed Target Site Selection

Orsolya Barabas, Donald R. Ronning, Catherine Guynet, Alison Burgess Hickman, Bao Ton-Hoang, Michael Chandler and Fred Dyda Cell 132: 208-220.[Full Text] [PDF]

The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.

16 . Chk1 Is a Histone H3 Threonine 11 Kinase that Regulates DNA Damage-Induced Transcriptional Repression

Midori Shimada, Hiroyuki Niida, Doaa H. Zineldeen, Hideaki Tagami, Masafumi Tanaka, Hiroyuki Saito and Makoto Nakanishi Cell 132: 221-232.[Full Text] [PDF]

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.

17 . Structure of a Sliding Clamp on DNA

Roxana E. Georgescu, Seung-Sup Kim, Olga Yurieva, John Kuriyan, Xiang-Peng Kong and Mike O'Donnell Cell 132: 43-54.[Full Text] [PDF]

The structure of the E. coli β clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the β ring. The pronounced 22° angle of DNA through β may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.

18 . A DNA Replication Mechanism for Generating Nonrecurrent Rearrangements Associated with Genomic Disorders

Jennifer A. Lee, Claudia M.B. Carvalho and James R. Lupski Cell 131: 1235-1247.[Full Text] [PDF]

The prevailing mechanism for recurrent and some nonrecurrent rearrangements causing genomic disorders is nonallelic homologous recombination (NAHR) between region-specific low-copy repeats (LCRs). For other nonrecurrent rearrangements, nonhomologous end joining (NHEJ) is implicated. Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused most frequently (60%–70%) by nonrecurrent duplication of the dosage-sensitive proteolipid protein 1 (PLP1) gene but also by nonrecurrent deletion or point mutations. Many PLP1 duplication junctions are refractory to breakpoint sequence analysis, an observation inconsistent with a simple recombination mechanism. Our current analysis of junction sequences in PMD patients confirms the occurrence of simple tandem PLP1 duplications but also uncovers evidence for sequence complexity at some junctions. These data are consistent with a replication-based mechanism that we term FoSTeS, for replication Fork Stalling and Template Switching. We propose that complex duplication and deletion rearrangements associated with PMD, and potentially other nonrecurrent rearrangements, may be explained by this replication-based mechanism.

19 . Tel2 Regulates the Stability of PI3K-Related Protein Kinases

Hiroyuki Takai, Richard C. Wang, Kaori K. Takai, Haijuan Yang and Titia de Lange Cell 131: 1248-1259.[Full Text] [PDF]

We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.

20 . CENP-B Controls Centromere Formation Depending on the Chromatin Context

Teruaki Okada, Jun-ichirou Ohzeki, Megumi Nakano, Kinya Yoda, William R. Brinkley, Vladimir Larionov and Hiroshi Masumoto Cell 131: 1287-1300.[Full Text] [PDF]

The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.