Christophe K. Mapendano

RNA Exosome Depletion Reveals Transcription Upstream of Active Human Promoters

Studies have shown that the bulk of eukaryotic genomes is transcribed. Transcriptome maps are frequently updated, but low-abundant transcripts have probably gone unnoticed. To eliminate RNA degradation, we depleted the exonucleolytic RNA exosome from human cells and then subjected the RNA to tiling microarray analysis. This revealed a class of short, polyadenylated and highly unstable RNAs. These promoter upstream transcripts (PROMPTs) are produced ∼0.5 to 2.5 kilobases upstream of active transcription start sites. PROMPT transcription occurs in both sense and antisense directions with respect to the downstream gene. In addition, it requires the presence of the gene promoter and is positively correlated with gene activity. We propose that PROMPT transcription is a common characteristic of RNA polymerase II (RNAPII) transcribed genes with a possible regulatory potential.

A SNP in the ABCC11 gene is the determinant of human earwax type

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G → A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G → A SNP is the first example of DNA polymorphism determining a visible genetic trait.

PROMoter uPstream Transcripts share characteristics with mRNAs and are produced upstream of all three major types of mammalian promoters

PROMoter uPstream Transcripts (PROMPTs) were identified as a new class of human RNAs, which are heterologous in length and produced only upstream of the promoters of active protein-coding genes. Here, we show that PROMPTs carry 3′-adenosine tails and 5′-cap structures. However, unlike mRNAs, PROMPTs are largely nuclear and rapidly turned over by the RNA exosome. PROMPT-transcribing DNA is occupied by RNA polymerase II (RNAPII) complexes with serine 2 phosphorylated C-terminal domains (CTDs), mimicking that of the associated genic region. Thus, the inefficient elongation capacity of PROMPT transcription cannot solely be assigned to poor CTD phosphorylation. Conditions that reduce gene transcription increase RNAPII occupancy of the upstream PROMPT region, suggesting that they reside in a common transcription compartment. Surprisingly, gene promoters that are actively transcribed by RNAPI or RNAPIII also produce PROMPTs that are targeted by the exosome. RNAPIII PROMPTs bear hallmarks of RNAPII promoter-associated RNAs, explaining the physical presence of RNAPII upstream of many RNAPIII-transcribed genes. We propose that RNAPII activity upstream gene promoters are wide-spread and integral to the act of gene transcription.

Role of DNA methylation and histone H3 lysine 27 methylation in tissue-specific imprinting of mouse Grb10.

ABSTRACT Mouse Grb10 is a tissue-specific imprinted gene with promoter-specific expression. In most tissues, Grb10 is expressed exclusively from the major-type promoter of the maternal allele, whereas in the brain, it is expressed predominantly from the brain type promoter of the paternal allele. Such reciprocally imprinted expression in the brain and other tissues is thought to be regulated by DNA methylation and the Polycomb group (PcG) protein Eed. To investigate how DNA methylation and chromatin remodeling by PcG proteins coordinate tissue-specific imprinting of Grb10, we analyzed epigenetic modifications associated with Grb10 expression in cultured brain cells. Reverse transcriptase PCR analysis revealed that the imprinted paternal expression of Grb10 in the brain implied neuron-specific and developmental stage-specific expression from the paternal brain type promoter, whereas in glial cells and fibroblasts, Grb10 was reciprocally expressed from the maternal major-type promoter. The cell-specific imprinted expression was not directly related to allele-specific DNA methylation in the promoters because the major-type promoter remained biallelically hypomethylated regardless of its activity, whereas gametic DNA methylation in the brain type promoter was maintained during differentiation. Histone modification analysis showed that allelic methylation of histone H3 lysine 4 and H3 lysine 9 were associated with gametic DNA methylation in the brain type promoter, whereas that of H3 lysine 27 regulated by the Eed PcG complex was detected in the paternal major-type promoter, corresponding to its allele-specific silencing. Here, we propose a molecular model that gametic DNA methylation and chromatin remodeling by PcG proteins during cell differentiation cause tissue-specific imprinting in embryonic tissues.

An ending is a new beginning: transcription termination supports re-initiation.

Comment on: Mapendano CK, et al. Mol Cell 2010; 40:410-22.

Expression of the Snurf-Snrpn IC transcript in the oocyte and its putative role in the imprinting establishment of the mouse 7C imprinting domain

AbstractThe human chromosome 15q11-q13, or mouse chromosome 7C, is an imprinting domain controlled by bipartite imprinting centers (ICs): Prader-Willi syndrome (PWS)-IC and Angelman syndrome (AS)-IC. PWS-IC functions to maintain the paternal epigenotype on the paternal chromosome in somatic cells, while AS-IC plays a role in the establishment of the maternal epigenetic mark at PWS-IC in the female germline or early embryos. Several alternative exons and promoters of Snurf-Snrpn (SNRPN upstream reading frame-small nuclear ribonucleoprotein polypeptide N) are expressed as “IC transcripts”. Previous studies have shown that IC-transcript expression is restricted to the brain. We studied expression of the mouse IC-transcript in tissues including brain and oocytes as well as in cultured neurons and glia cells by RT-PCR and by in situ hybridization (ISH) in oocytes. The IC transcript was strongly expressed in brain (especially in neurons) and ovary (especially in oocytes and granulosa cells), while no expression was found in other tissues. This was confirmed by quantitative analysis and ISH. Expression levels in the brain were 7-fold higher compared to those in ovaries. ISH signals were observed in oocytes and granulosa cells of the secondary and developing follicles. These findings, together with previous data, suggest that the IC transcript may be associated with the establishment of PWS-IC methylation on the maternal chromosome as an AS-IC cis-acting element.

Relationships between PROMPT and gene expression

ABSTRACT Most mammalian protein-coding gene promoters are divergent, yielding promoter upstream transcripts (PROMPTs) in the reverse direction from their conventionally produced mRNAs. PROMPTs are rapidly degraded by the RNA exosome rendering a general function of these molecules elusive. Yet, levels of certain PROMPTs are altered in stress conditions, like the DNA damage response (DDR), suggesting a possible regulatory role for at least a subset of these molecules. Here we manipulate PROMPT levels by either exosome depletion or UV treatment and analyze possible effects on their neighboring genes. For the CTSZ and DAP genes we find that TFIIB and TBP promoter binding decrease when PROMPTs accumulate. Moreover, DNA methylation increases concomitant with the recruitment of the DNA methyltransferase DNMT3B. Thus, although a correlation between increased PROMPT levels and decreased gene activity is generally absent, some promoters may have co-opted their divergent transcript production for regulatory purposes.

Comprehensive multiregional analysis of molecular heterogeneity in bladder cancer

Genetic alterations identified in adjacent normal appearing tissue in bladder cancer patients are indicative of a field disease. Here we assessed normal urothelium transformation and intra-tumour heterogeneity (ITH) in four patients with bladder cancer. Exome sequencing identified private acquired mutations in a lymph node metastasis and local recurrences. Deep re-sequencing revealed presence of at least three and four subclones in two patients with multifocal disease, while no demarcation of subclones was identified in the two patients with unifocal disease. Analysis of adjacent normal urothelium showed low frequency mutations in patients with multifocal disease. Expression profiling showed intra-tumour and intra-patient co-existence of basal- and luminal-like tumour regions, and patients with multifocal disease had a greater degree of genomic and transcriptomic ITH, as well as transformation of adjacent normal cells, compared to patients with unifocal disease. Analysis of the adjacent urothelium may pave the way for therapies targeting the field disease.