rDNA Clusters Make Contact with Genes that Are Involved in Differentiation and Cancer and Change Contacts after Heat Shock Treatment

Human rDNA clusters form numerous contacts with different chromosomal regions as evidenced by chromosome conformation capture data. Heterochromatization of rDNA genes leads to heterochromatization in different chromosomal regions coupled with the activation of the transcription of genes related to differentiation. These data suggest a role for rDNA clusters in the regulation of many human genes. However, the genes that reside within the rDNA-contacting regions have not been identified. The purpose of this study was to detect and characterize the regions where rDNA clusters make frequent contacts and to identify and categorize genes located in these regions. We analyzed the regions that contact rDNA using 4C data and show that these regions are enriched with genes specifying transcription factors and non-coding RNAs involved in differentiation and development. The rDNA-contacting genes are involved in neuronal development and are associated with different cancers. Heat shock treatment led to dramatic changes in the pattern of rDNA-contacting sites, especially in the regions possessing long stretches of H3K27ac marks. Whole-genome analysis of rDNA-contacting sites revealed specific epigenetic marks and the transcription sites of 20–100 nt non-coding RNAs in these regions. The rDNA-contacting genes jointly regulate many genes that are involved in the control of transcription by RNA polymerase II and the development of neurons. Our data suggest a role for rDNA clusters in the differentiation of human cells and carcinogenesis

Dynamics of Whole-Genome Contacts of Nucleoli in Drosophila Cells Suggests a Role for rDNA Genes in Global Epigenetic Regulation

Chromosomes are organized into 3D structures that are important for the regulation of gene expression and differentiation. Important role in formation of inter-chromosome contacts play rDNA clusters that make up nucleoli. In the course of differentiation, heterochromatization of rDNA units in mouse cells is coupled with the repression or activation of different genes. Furthermore, the nucleoli of human cells shape the direct contacts with genes that are involved in differentiation and cancer. Here, we identified and categorized the genes located in the regions where rDNA clusters make frequent contacts. Using a 4C approach, we demonstrate that in Drosophila S2 cells, rDNA clusters form contacts with genes that are involved in chromosome organization and differentiation. Heat shock treatment induces changes in the contacts between nucleoli and hundreds of genes controlling morphogenesis. We show that nucleoli form contacts with regions that are enriched with active or repressive histone marks and where small non-coding RNAs are mapped. These data indicate that rDNA contacts are involved in the repression and activation of gene expression and that rDNA clusters orchestrate large groups of Drosophila genes involved in differentiation

Six Highly Conserved Targets of RNAi Revealed in HIV-1-Infected Patients from Russia Are Also Present in Many HIV-1 Strains Worldwide

RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%–97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Keywords: HIV-1, RNAi targets, gene therapy, ultra-deep sequencing, conserved HIV-1 sequence

DNA double-strand breaks coupled with PARP1 and HNRNPA2B1 binding sites flank coordinately expressed domains in human chromosomes.

Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs) is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50-250 kb DNA domains. We found that about 30% of the domains (denoted forum domains) possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites

Selection of proteins binding with RAFT preparations and with the individual FT from <i>WWOX</i> gene.

<p>All indicated proteins were identified by mass spectrometry as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003429#pgen.1003429.s019" target="_blank">Text S1</a>. (A) Binding of nuclear proteins with biotinylated RAFT preparations (0.4 µg) was revealed by the use of SA-PMP (see Extended Experimental Procedures in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003429#pgen.1003429.s019" target="_blank">Text S1</a>). Poly[d(I)/d(C)] competitor DNA (dI/dC) and poly[(I)/(C)] competitor RNA (I/C) were used. E, extract of nuclear proteins; M, marker. Proteins were separated by use of 5% PAGE. (B) Binding of nuclear proteins to biotinylated RAFT preparations. dI/dC and I/C non-specific competitors, and PCR-amplified non-specific competitor and RAFT-specific competitor DNAs, both synthesized using Taq polymerase, were used. The non-specific DNA competitor efficiently eliminates end-binding proteins. E, extract of nuclear proteins; M, marker. Lanes 4 and 5 correspond to experiments with single-stranded or double-stranded biotinylated oligos, respectively, that were used for amplification of the RAFT probes. Proteins were separated by use of 5–18% PAGE. (C) Binding of nuclear proteins to biotinylated 1050-bp <i>WWOX</i> FT preparations (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003429#pgen.1003429.s004" target="_blank">Figure S4</a>). dI/dC and I/C non-specific competitors, and PCR-amplified non-specific competitor and <i>WWOX-</i>specific competitor DNAs, both synthesized using Taq polymerase, were used. E, extract of nuclear proteins; M, marker. Proteins were separated by use of 5–18% PAGE. (D) Binding of nuclear proteins to biotinylated RAFT preparations. dI/dC and I/C non-specific competitors, and PCR-amplified non-specific competitor and RAFT-specific competitor DNAs, both synthesized using Taq polymerase, were used. Lanes 1 and 3 correspond to experiments with 20x excesses of RAFT preparation lacking the biotin label (8 µg) or total human DNA (8 µg) digested with Sau3A enzyme, respectively (competitors). Lane 2 corresponds to the experiment with no specific competitors; M, marker. Proteins were separated by use of 5% PAGE.</p

FT density across chromosomes 2, 3, 6, 7, 16, and X, which possess the frequently and less frequently observed CFS in leukocytes.

<p>Window = 500 kb; step = 100 kb. The frequently and less frequently observed CFS detected in leukocytes are shown in red and in blue, respectively.</p

ChIP experiments using antibodies to PARP1 or to HNRNPA2B1.

<p>(A) Scheme illustrating the PCR strategies used for amplification of DNA fragments across FT or around it. (B) Results of PCR across four FT and two non-FT regions (from the <i>WWOX</i> gene and the 5.8S ribosomal gene) using immunoprecipitated DNA. Primers used are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003429#pgen.1003429.s016" target="_blank">Table S1</a>. Percentage of input DNA is indicated, n = 4. (C) Results of PCR around two FT. Primers used are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003429#pgen.1003429.s017" target="_blank">Table S2</a>. Percentage of input DNA is indicated, n = 4.</p

Coordinated expression inside forum domains.

<p>The UCSC Genome Browser, Human Feb. 2009 (GRCh37/hg19) Assembly was used. UCSC genes, human mRNAs from GenBank, the H3K37Ac mark from Encode, chromatin state segmentation by HMM from Encode/Broad, and some histone modifications by ChIP-seq from Encode are indicated (Ernst et al., 2011). The “RNA-seq” lane corresponds to the expression of mRNAs in IMR90 cells (GEO accession number GSM438363). The “Domains” lanes indicate the forum domains containing silent or weakly expressed genes (blue brackets), or domains possessing actively transcribed genes (red brackets). “HEK293T” lanes correspond to the expression of mRNA in HEK293T cells (microarray data using Affymetrix Human Exon 1.0 ST expression arrays, <a href="http://www.affymetrix.com/estore/browse/products.jsp?navMode=34000&productId=131452&navAction=jump&aId=productsNav#1_1" target="_blank">http://www.affymetrix.com/estore/browse/products.jsp?navMode=34000&productId=131452&navAction=jump&aId=productsNav#1_1</a>, wgEncodeEH002692_2). (A) Region of chr17 that includes the <i>HOXB</i> gene cluster and contains active and silent domains. (B) Region of chr12 that includes active, low expressing, and silent domains. The leftmost domains are actively transcribed in IMR90 cells, but are low expressed in HEK 293T cells. (C) Region of chr16 that includes active and silent domains in IMR90 cells, and active and low expressing domains in HEK293T cells.</p

Overviews of chromosomes 3 and 16, which contain the FRA3B and FRA16D regions.

<p>The frequently and less frequently observed CFS detected in leukocytes are shown in red and blue, respectively. The Integrated Genome Browser (Affymetrix) was used (<a href="http://bioviz.org/igb/" target="_blank">http://bioviz.org/igb/</a>). (A) FT barcode in the overview of chr3. (B) FRA3B region that contains the <i>FHIT</i> gene. Mapped FT are shown. The length and number of reads are indicated. Blue arrows indicate the positions of the previously described DNA breaks inside <i>FHIT</i> in tumor cells (GenBank: AF020610.1 and U85047.1). (C) FT barcode in the overview of chr16. (D) FRA16D region containing the <i>WWOX</i> gene. Mapped FT are shown. The length and number of reads are indicated. The previously mapped breaks inside the minisatellite repeat in the <i>WWOX</i> gene are indicated by the red arrow (GenBank: U85253.1). (E) The length and number of FT reads inside the 3′ exon are indicated. (F) Mapped cut sites inside the 3′ exon are indicated by black arrows. Some of the corresponding RAFT reads are shown at the bottom.</p

Fluorescent <i>in situ</i> hybridization of the RAFT probe.

<p>(A) Hybridization patterns of the RAFT probe and total DNA. (B) Hybridization patterns inside chr2 shown in more detail.</p