Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

ASAR15, A cis-acting locus that controls chromosome-wide replication timing and stability of human chromosome 15.

DNA replication initiates at multiple sites along each mammalian chromosome at different times during each S phase, following a temporal replication program. We have used a Cre/loxP-based strategy to identify cis-acting elements that control this replication-timing program on individual human chromosomes. In this report, we show that rearrangements at a complex locus at chromosome 15q24.3 result in delayed replication and structural instability of human chromosome 15. Characterization of this locus identified long, RNA transcripts that are retained in the nucleus and form a "cloud" on one homolog of chromosome 15. We also found that this locus displays asynchronous replication that is coordinated with other random monoallelic genes on chromosome 15. We have named this locus ASynchronous replication and Autosomal RNA on chromosome 15, or ASAR15. Previously, we found that disruption of the ASAR6 lincRNA gene results in delayed replication, delayed mitotic condensation and structural instability of human chromosome 6. Previous studies in the mouse found that deletion of the Xist gene, from the X chromosome in adult somatic cells, results in a delayed replication and instability phenotype that is indistinguishable from the phenotype caused by disruption of either ASAR6 or ASAR15. In addition, delayed replication and chromosome instability were detected following structural rearrangement of many different human or mouse chromosomes. These observations suggest that all mammalian chromosomes contain similar cis-acting loci. Thus, under this scenario, all mammalian chromosomes contain four distinct types of essential cis-acting elements: origins, telomeres, centromeres and "inactivation/stability centers", all functioning to promote proper replication, segregation and structural stability of each chromosome

Differential Allelic Expression among Long Non-Coding RNAs

Long non-coding RNAs (lncRNA) comprise a diverse group of non-protein-coding RNAs >200 bp in length that are involved in various normal cellular processes and disease states, and can affect coding gene expression through mechanisms in cis or in trans. Since the discovery of the first functional lncRNAs transcribed by RNA Polymerase II, H19 and Xist, many others have been identified and noted for their unusual transcriptional pattern, whereby expression from one chromosome homolog is strongly favored over the other, also known as mono-allelic or differential allelic expression. lncRNAs with differential allelic expression have been observed to play critical roles in developmental gene regulation, chromosome structure, and disease. Here, we will focus on known examples of differential allelic expression of lncRNAs and highlight recent research describing functional lncRNAs expressed from both imprinted and random mono-allelic expression domains

DRT/DMC on an engineered t (15;16).

<p>A) Diagram of the Aprt-loxP cassettes, and the genomic organization of the mouse <i>Aprt</i> gene is shown. The 5′ portion of the Aprt gene was separated from the 3′ portion to generate the AP-loxP and loxP-RT cassettes. Each cassette contains a loxP site in the second intron of the Aprt gene, and each cassette was cloned separately into Lentiviral vectors. B) Illustration of the original loxP cassette integration sites in chromosomes 15 (green) and 16 (red) in P268 cells, and the balanced translocation, t (15;16), generated in R268 cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004923#pgen.1004923-Breger2" target="_blank">[17]</a>). C and D) DMC on the chromosome 15 derivative of the t (15;16). R268 cells were harvested for mitotic cells, dropped on slides and processed for DNA FISH using chromosome 15 and 16 centromeric probes [CHR15 (green) and CHR16 (red)]. E) Diagram of chromosome 15 showing the orientation and integration site (∼76.86 mb) of the original loxP-3′RT cassette in P268 cells. P268 cells were infected with Lentiviral vectors containing either the AP-loxP or loxP-RT cassettes, and 18 pools of 5,000–10,000 infected clones isolated for each Lentivirus. The structure of the AP-loxP Lentivirus (in the opposite orientation; green), the extent of 5 distal deletions (Δ135 kb, Δ161 kb, Δ255 kb, Δ5.6 mb and Δ12.8 mb) and 2 inversions (I644 kb, and I785 kb), BACs (CTD-2117F7 and CTD-2299E17), and the protein-coding genes <i>ISL2</i>, <i>SCAPER, RCN2, PSTPIP1, TSPAN3</i> and <i>PEAK1</i> are indicated. The approximate location of the micro RNA gene <i>MIR3713</i> is shown with an asterisk.</p

DNA FISH analysis of human loci.

<p>The percentage of the single-double (%SD) pattern was determined using DNA FISH.</p><p>Coordinated asynchronous replication was scored against BAC CTD-2299E17.</p><p>DNA FISH analysis of human loci.</p

Coordinated random asynchronous replication on chromosome 15.

<p>A–C) ReTiSH assay on rDNA loci in PBLs. PBLs were labeled with BrdU for 14 (A) or 6 (B) hours, arrested in metaphase, and subjected to ReTiSH using an 18S rDNA probe (red). The chromosome 15 s were identified using a centromeric probe (green), and the chromosomal DNA was detected with DAPI. A and B) The DAPI images of the chromosomes were inverted and the banding patterns were used to identify all of the ReTiSH positive chromosomes. The arrows mark the chromosome 15 s, and the arrowheads mark the other four chromosomes containing rDNA clusters (13, 14, 21, and 22). C) The ReTiSH signals for the rDNA (red) and chromosome 15 centromeric (green) probes from the 14 (panel A) and 6 (panel B) hour time points are shown. The early and late replicating chromosome 15 s are indicated for the 6 hour time point. D) ReTiSH assay using an <i>ASAR15</i> BAC (CTD-2299E17; red), an rDNA probe (red), and a chromosome 15 centromeric probe (green). The <i>ASAR15</i> and rDNA probes show hybridization signals to the same chromosome 15 homolog at the 6 hour time point. E) ReTiSH assay using an <i>ASAR15</i> BAC (CTD-2299E17; red), a <i>MYO1E</i> BAC (RP11-1089J12; green) and a chromosome 15 centromeric probe (red). The <i>ASAR15</i> BAC and the <i>MYO1E</i> BACs show hybridization signals to the same chromosome 15 homolog at the 6 hour time point. D and E) The early and late replicating chromosome 15 s are indicated.</p

Chromosome rearrangements and delayed replication of a Cre/loxP-mediated deletion (∼135 kb) in chromosome 15.

<p>A) Secondary rearrangements of chromosome 15. Δ268-4f cells were processed for DNA FISH using a chromosome 15 WCP, and the DNA was stained with DAPI. Rearrangements involving chromosome 15 are indicated with arrows. Non-rearranged chromosome 15 s are indicated with asterisks. B) Schematic diagram of the BrdU Terminal Label replication-timing assay <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004923#pgen.1004923-Smith2" target="_blank">[23]</a>. Cells were exposed to BrdU for either 4.5 or 6 hours, harvested for mitotic cells, and processed for BrdU incorporation and DNA FISH to identify chromosome 15. C) BrdU-WCP assay on cells containing an ∼135 kb distal deletion in chromosome 15. Δ268-4f cells were exposed to BrdU for 4.5 hours, harvested for mitotic cells, stained with an anti-BrdU antibody (green), and processed for DNA FISH with a chromosome 15 WCP (CHR 15; red). The DNA was stained with DAPI. Two different chromosome 15 secondary rearrangements are indicated with arrows. The inset shows the derivative chromosome 15 with the asterisk, with the BrdU staining and WCP shown in separate images. The brackets highlight the non-chromosome 15 DNA. D–G) BrdU-BAC assay on cells containing an ∼135 kb distal deletion in chromosome 15. Δ268-4c cells were exposed to BrdU for 4.5 hours, harvested for mitotic cells, stained with an anti-BrdU antibody (green), and processed for DNA FISH with a chromosome 15 centromeric probe (red) plus a BAC (CTD-2299E17; red) from the deleted region. The DNA was stained with DAPI (white in panel D or blue in panels E–G). The arrows mark the centromeric signals, and the arrowheads mark the BAC signals. The asterisks mark short arms of the deleted chromosome 15 s, which contain BrdU incorporation.</p

Delayed replication of chromosome 15 with an Cre/loxP-mediated ∼161 kb distal deletion.

<p>A–F) Δ268-4 g cells were incubated with BrdU for 6 hours, harvested for mitotic cells, stained with an antibody to BrdU (green) and processed for DNA FISH using a chromosome 15 centromeric probe (red) plus BAC CTD-2299E17 (red). The chromosomal DNA was stained with DAPI (blue). A and B) A metaphase spread containing three chromosome 15 s (i, ii, and iii). C) The three chromosome 15 s from panel B were cut out and aligned showing the BrdU and FISH signals in separate images. The asterisk marks the location of the deletion in the chromosome marked i, and the arrows mark the location of the BAC hybridization signals on chromosomes ii and iii. D) Pixel intensity profiles of the BrdU incorporation (green), and DAPI (blue) staining along the three chromosome 15 s from panel B. E) The pixel intensity (average intensity x area) for each chromosome, i, ii, and iii, showing the total amount of BrdU incorporation or DAPI staining. F) Quantification of the BrdU incorporation in multiple cells. The red and blue bars represent deleted and non-deleted chromosome 15 s, respectively, in 7 different cells. G) Instability of chromosome 15 containing an ∼161 kb Cre-loxP deletion. Mitotic Δ268-4e cells were processed for DNA FISH with a chromosome 15 WCP, and the chromosomal DNA was stained with DAPI. Rearrangements involving chromosome 15 are indicated with arrows, and non-rearranged chromosome 15 s are indicated with asterisks.</p

Cre/loxP-mediated chromosome 15 rearrangements.

#<p>The number of clones that showed DRT/DMC (total clones scored).</p>q<p>The number of clones that showed>10% of rearrangements of chromosome 15 (total clones scored).</p><p>*At least one clone with>90% of cells containing chromosome 15 rearrangements.</p><p>Cre/loxP-mediated chromosome 15 rearrangements.</p

The Cre/loxP-mediated deletions occurred on the expressed allele of <i>ASAR15</i>.

<p>P268 (panel A) and Δ268-4 g (panel B) cells were subjected to RNA FISH using the H1 (green) and E4 (red) probes to detect RNA. Images of the RNA hybridization signals were obtained, and the coordinates of individual cells were recorded; the slides were subsequently processed for DNA FISH (BACs CTD-2299E17 plus BAC-CTD-2117F7) and new images of the DNA hybridization were captured for the same cells. The DNA FISH step included an RNAase step, which eliminated the RNA FISH signals. The DNA FISH hybridization signal was pseudo-colored purple for clarity, and the nuclear DNA was stained with DAPI (blue). Representative images from three different P268 and Δ268-4 g cells (#1–3) are shown in each panel (A and B). The arrowheads mark the coincident sites of hybridization detected by both probes. The arrows mark the sites of hybridization with the H1 probe that was not coincident with a site of hybridization with the E4 probe in Δ268-4 g cells (panel B). The asterisks mark the sites of DNA hybridization that lacked corresponding RNA hybridization signals from either H1 or E4 probes.</p