Roche NimbleGen Webinar Series

Large Noncoding RNAs (ncRNA) are becoming a distinguishing feature of the Metazoan genomes, but their functional roles are poorly understood. Here we describe a novel type of ncRNA termed HOTAIR that is 2.2. Kb RNA, has 5 spliced exons, a poly A tail and a 5’ meC cap, yet has no potential to code a sensible amino-acid sequence. HOTAIR is encoded antisense to the human HOXC cluster at the exact juncture of a 40 Kb domain of heterochromatin and a 60 Kb domain of euchromatin. However, HOTAIR doesn’t serve to regulate this boundary; Remarkably HOTAIR affects the global epigenetic state of the HOXD cluster located on a separate chromosome. HOTAIR binds the Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of large ncRNA may demarcate chromosomal domains of gene silencing from a distance. We further discuss RNA labeling optimizations, platform comparisons and the integration of ChIP-Chip and RNA expression on high-resolution DNA tiling arrays that were critical for our discovery of HOTAIR. Together, these results demonstrate the power of integrative genomics in elucidating the biological roles of large ncRNAs.

Microarray analysis is a powerful tool for quantifying genome-wide changes in gene expression. To facilitate wider application of microarray data in biomedical research and promote the use of microarray analysis in diagnostic and regulatory fields, it is important that data obtained using this technology is accurate and reliable. As such, we evaluated the performance of NimbleGen gene expression microarrays using standard RNA samples recently used by the Microarray Quality Control Consortium, a project established to evaluate the reproducibility and quality of data obtained using microarrays from multiple suppliers. Here we show high inter- array reproducibility was achieved for both the 1-plex and 4-plex expression microarrays, demonstrating the reliability of NimbleChip microarray data. We also show a high concordance between data obtained from NimbleChip microarray analysis and data from TaqMan analysis, the current gold standard for mRNA level quantitation, indicating the accuracy of NimbleChip microarray data in assessing gene expression values.

Microarray analysis is a powerful tool for quantifying genome-wide changes in gene expression. To facilitate wider application of microarray data in biomedical research and promote the use of microarray analysis in diagnostic and regulatory fields, it is important that data obtained using this technology is accurate and reliable. As such, we evaluated the performance of NimbleGen gene expression microarrays using standard RNA samples recently used by the Microarray Quality Control Consortium, a project established to evaluate the reproducibility and quality of data obtained using microarrays from multiple suppliers. Here we show high inter- array reproducibility was achieved for both the 1-plex and 4-plex expression microarrays, demonstrating the reliability of NimbleChip microarray data. We also show a high concordance between data obtained from NimbleChip microarray analysis and data from TaqMan analysis, the current gold standard for mRNA level quantitation, indicating the accuracy of NimbleChip microarray data in assessing gene expression values.

Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.

Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.

DNA methylation at cytosines residues can mediate epigenetic gene silencing and is often perturbed in cancer cells. To gain insight into the function of DNA methylation at promoters and its impact on gene expression, we measure DNA methylation at all human promoters using Methylated DNA Immunoprecipitation (MeDIP) coupled with high density oligonucleotide arrays. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes gain promoter DNA methylation during somatic development, suggesting additional functional selection. These results show that promoter structure and gene function are major predictors of DNA methylation states.

Audio: NimbleGen ESHG 2007 Workshop - Full Program

Video: NimbleGen ESHG 2007 Workshop - Full Program

Copy number variation (CNV) in the genome is extensive and yet is grossly under-ascertained. The resolution of CNV detection of most current technology platforms is approximately 50kb, and yet copy number variation two orders of magnitude smaller than this is likely to go undetected by exon resequencing. Furthermore, we know that smaller CNVs are far more numerous than larger CNVs, and so improved CNV detection resolution can be expected to dramatically increase the numbers of known CNVs. We present preliminary data from our project to detect all common CNVs (with minor allele frequencies of 5% of more) of length 500bp or greater by performing array-based comparative genome hybridisation on oligonucleotide arrays that tile across the assayable portions of the human genome.

Copy number variation (CNV) in the genome is extensive and yet is grossly under-ascertained. The resolution of CNV detection of most current technology platforms is approximately 50kb, and yet copy number variation two orders of magnitude smaller than this is likely to go undetected by exon resequencing. Furthermore, we know that smaller CNVs are far more numerous than larger CNVs, and so improved CNV detection resolution can be expected to dramatically increase the numbers of known CNVs. We present preliminary data from our project to detect all common CNVs (with minor allele frequencies of 5% of more) of length 500bp or greater by performing array-based comparative genome hybridisation on oligonucleotide arrays that tile across the assayable portions of the human genome.

DNA methylation at cytosines residues can mediate epigenetic gene silencing and is often perturbed in cancer cells. To gain insight into the function of DNA methylation at promoters and its impact on gene expression, we measure DNA methylation at all human promoters using Methylated DNA Immunoprecipitation (MeDIP) coupled with high density oligonucleotide arrays. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes gain promoter DNA methylation during somatic development, suggesting additional functional selection. These results show that promoter structure and gene function are major predictors of DNA methylation states.

A single RNA polymerase is responsible for all transcription in the bacterium Escherichia coli. We have investigated how the genome-wide distribution of this RNA polymerase relates to the level of transcription. Using ChIP-chip we determined the genome-wide association of RNA polymerase. We also determined an unbiased transcript map from the same cells. We identified many novel, non-coding RNAs, including intergenic and intragenic sense and antisense RNAs. Interestingly, there is not a perfect relationship between the distribution of RNA polymerase and the level of transcription. At most transcribed regions RNA polymerase associates with promoters at a much higher level than with coding sequences. Strikingly, almost a quarter of all promoters that bind RNA polymerase are not detectably transcribed. We propose that RNA polymerase is "poised" at these promoters, ready to transcribe the corresponding gene under the appropriate environmental condition.

A single RNA polymerase is responsible for all transcription in the bacterium Escherichia coli. We have investigated how the genome-wide distribution of this RNA polymerase relates to the level of transcription. Using ChIP-chip we determined the genome-wide association of RNA polymerase. We also determined an unbiased transcript map from the same cells. We identified many novel, non-coding RNAs, including intergenic and intragenic sense and antisense RNAs. Interestingly, there is not a perfect relationship between the distribution of RNA polymerase and the level of transcription. At most transcribed regions RNA polymerase associates with promoters at a much higher level than with coding sequences. Strikingly, almost a quarter of all promoters that bind RNA polymerase are not detectably transcribed. We propose that RNA polymerase is "poised" at these promoters, ready to transcribe the corresponding gene under the appropriate environmental condition.

We are interested in the genetic underpinnings of behavioral disorders in children. We describe three families with recurrent deletions affecting a region of chromosome 10q that produces a range of language and behavioral deficits. These deletions are in a region with a high density of large low copy repeats (LCRs), suggesting these features of genomic architecture are involved in the production and frequency of these chromosome rearrangements. We have employed NimbleGen oligonucleotide arrays to fine-map these deletions, even those located within LCR elements. Affected genes in this interval include NRG3, GRID1 and PTEN, a tumor suppressor and lipid phosphatase. PTEN mutations have been associated with a subset of autistic children, suggesting signaling regulated by this growth regulator may be involved in autism spectrum disorder.

We are interested in the genetic underpinnings of behavioral disorders in children. We describe three families with recurrent deletions affecting a region of chromosome 10q that produces a range of language and behavioral deficits. These deletions are in a region with a high density of large low copy repeats (LCRs), suggesting these features of genomic architecture are involved in the production and frequency of these chromosome rearrangements. We have employed NimbleGen oligonucleotide arrays to fine-map these deletions, even those located within LCR elements. Affected genes in this interval include NRG3, GRID1 and PTEN, a tumor suppressor and lipid phosphatase. PTEN mutations have been associated with a subset of autistic children, suggesting signaling regulated by this growth regulator may be involved in autism spectrum disorder.