Evidence for RNA synthesis in the intergenic region between enhancer and promoter and its inhibition by insulators in Drosophila melanogaster

Uncovering the nature of communication between enhancers, promoters and insulators is important for understanding the fundamental mechanisms that ensure appropriate gene expression levels. Here we describe an approach employing transient expression of genetic luciferase reporter gene constructs with quantitative RT–PCR analysis of transcription between an enhancer and Hsp70 promoter. We tested genetic constructs containing gypsy and/or Fab7 insulators in different orientations, and an enhancer from copia LTR-retroelement [(enh)copia]. A single gypsy or Fab7 insulator inserted between the promoter and enhancer in any polarity reduced enhancer action. A pair of insulators flanking the gene in any orientation exhibited increased insulation activity. We detected promoter-independent synthesis of non-coding RNA in the intergenic region of the constructs, which was induced by the enhancer in both directions and repressed by a single insulator or a pair of insulators. These results highlight the involvement of RNA-tracking mechanisms in the communications between enhancers and promoters, which are inhibited by insulators

Genome-wide profiling of forum domains in Drosophila melanogaster

Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Most forum domains are 50–200 kb in length. We mapped forum domain termini using FISH on polytene chromosomes and we performed genome-wide mapping using a Drosophila melanogaster genomic tiling microarray consisting of overlapping 3 kb fragments. We found that forum termini very often correspond to regions of intercalary heterochromatin and regions of late replication in polytene chromosomes. We found that forum domains contain clusters of several or many genes. The largest forum domains correspond to the main clusters of homeotic genes inside BX-C and ANTP-C, cluster of histone genes and clusters of piRNAs. PRE/TRE and transcription factor binding sites often reside inside domains and do not overlap with forum domain termini. We also found that about 20% of forum domain termini correspond to small chromosomal regions where Ago1, Ago2, small RNAs and repressive chromatin structures are detected. Our results indicate that forum domains correspond to big multi-gene chromosomal units, some of which could be coordinately expressed. The data on the global mapping of forum domains revealed a strong correlation between fragmentation sites in chromosomes, particular sets of mobile elements and regions of intercalary heterochromatin

by insulators in

Evidence for RNA synthesis in the intergenic regio

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

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

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

Expression levels inside forum domains in chr1, chr2, chr3, and chr4.

<p>The data for expression 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) were used. The median values of transcription levels in coding regions (representing exon array signals) within a particular forum domain were used, and the result was plotted according to the position of the domain in its chromosome. (A–D) Expression levels inside forum domains in the largest chr1, chr2, chr3, and chr4. The arrows indicate the position of the average expression level of forum domains in a particular chromosome. The value to the right of the arrow indicates the proportion of forum domains in a chromosome that is more highly expressed. Domains with expression levels lower or higher than the average expression level are shown by the blue and red spots, respectively.</p