Developmental control of gene copy number by repression of replication initiation and fork progression

Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
Genome Research (Impact Factor: 14.63). 11/2011; 22(1):64-75. DOI: 10.1101/gr.126003.111
Source: PubMed


Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

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Available from: Matthew Eaton, Sep 23, 2014
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    • "We also found (Table 1; Supplemental Fig. S1), as previously reported for strong UR regions (Sher et al. 2012), that virtually all of the UR regions corresponded closely to domains of repressive chromatin as defined in genomic studies (Karchenko et al. 2012). To investigate when underreplication occurs during development, we analyzed second instar larval (L2) salivary glands, which have completed about seven endocycles. "
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    ABSTRACT: DNA replication remains unfinished in many Drosophila polyploid cells, which harbor disproportionately fewer copies of late-replicating chromosomal regions. By analyzing paired-end high-throughput sequence data from polytene larval salivary gland cells, we define 112 underreplicated (UR) euchromatic regions 60-480 kb in size. To determine the effects of underreplication on genome integrity, we analyzed anomalous read pairs and breakpoint reads throughout the euchromatic genome. Each UR euchromatic region contains many different deletions 10-500 kb in size, while very few deletions are present in fully replicated chromosome regions or UR zones from embryo DNA. Thus, during endocycles, stalled forks within UR regions break and undergo local repair instead of remaining stable and generating nested forks. As a result, each salivary gland cell contains hundreds of unique deletions that account for their copy number reductions. Similar UR regions and deletions were observed in ovarian DNA, suggesting that incomplete replication, fork breakage, and repair occur widely in polytene cells. UR regions are enriched in genes encoding immunoglobulin superfamily proteins and contain many neurally expressed and homeotic genes. We suggest that the extensive somatic DNA instability described here underlies position effect variegation, molds the structure of polytene chromosomes, and should be investigated for possible functions.
    Genes & Development 08/2014; 28(16):1840-55. DOI:10.1101/gad.245811.114 · 10.80 Impact Factor
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    • "I - DNAse I hypersensitivity sites (high magnitude DHS - vertical lines) in S2, BG3, and Kc cells by Kharchenko et al. [22]. J - ORC2-binding sites in S2, BG3, Kc cells and salivary glands by Eaton et al. [45], Sher et al. [46]. K – Enrichment profiles of NSL complex components: NSL1 binding profile from salivary glands by Raja et al. [47], NSL3 in S2 cells by Lam et al. [48], NSL1 in S2 cells by Feller et al. [49]. "
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    ABSTRACT: Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential "head-to-head" orientation of genes. They are enriched for "broad" class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called "grey" bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.
    PLoS ONE 07/2014; 9(7):e101631. DOI:10.1371/journal.pone.0101631 · 3.23 Impact Factor
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    • "This might seem unlikely, since partially rereplicated DNA can lead to gene amplification (Green et al., 2010), but it would be preferable to having unrestrained forks, and rereplicated DNA could be subjected to subsequent repair by nascent strand displacement or exonuclease activity . A precedent for this kind of mechanism exists in Drosophila polytene chromosomes, in which the SUUR protein represses fork movement, possibly via an effect on chromatin structure involving the repressive H3K27me3 modification (Sher et al., 2012). Maintaining genome stability in gamete formation is important, particularly in multicellular organisms, which may have hundreds of thousands of replication origins, and high-fidelity licensing control may fail to block completely illegal initiation events. "
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    ABSTRACT: Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin dependent kinase activity, activation of Dbf4-dependent kinase or deletion of the Spd1-inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I-II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.
    Molecular biology of the cell 01/2013; 24(5). DOI:10.1091/mbc.E12-11-0825 · 4.47 Impact Factor
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