The ability to quantitatively survey the global behavior of transcriptomes has been a key milestone in the field of systems biology, enabled by the advent of DNA microarrays. While this approach has ...literally transformed our vision and approach to cellular physiology, microarray technology has always been limited by the requirement to decide,
a priori, what regions of the genome to examine. While very high density tiling arrays have reduced this limitation for simpler organisms, it remains an obstacle for larger, more complex, eukaryotic genomes.
The recent development of “next-generation” massively parallel sequencing (MPS) technologies by companies such as Roche (454 GS FLX), Illumina (Genome Analyzer II), and ABI (AB SOLiD) has completely transformed the way in which quantitative transcriptomics can be done. These new technologies have reduced both the cost-per-reaction and time required by orders of magnitude, making the use of sequencing a cost-effective option for many experimental approaches. One such method that has recently been developed uses MPS technology to directly survey the RNA content of cells, without requiring any of the traditional cloning associated with EST sequencing. This approach, called “RNA-seq”, can generate quantitative expression scores that are comparable to microarrays, with the added benefit that the entire transcriptome is surveyed without the requirement of
a priori knowledge of transcribed regions. The important advantage of this technique is that not only can quantitative expression measures be made, but transcript structures including alternatively spliced transcript isoforms, can also be identified. This article discusses the experimental approach for both sample preparation and data analysis for the technique of RNA-seq.
Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and that many functional properties of transcripts are based not on ...coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or few experimental conditions. Here we applied direct high-throughput sequencing of complementary DNAs (RNA-Seq), supplemented with data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including rapid proliferation, meiotic differentiation and environmental stress, as well as in RNA processing mutants to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to sample transcriptomes deeply at maximal resolution. In contrast to hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not robustly transcribed or rapidly degraded. The combined sequencing and strand-specific array data provide rich condition-specific information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus improving the existing genome annotation. Sequence reads spanning exon-exon or exon-intron junctions give unique insight into a surprising variability in splicing efficiency across introns, genes and conditions. Splicing efficiency was largely coordinated with transcript levels, and increased transcription led to increased splicing in test genes. Hundreds of introns showed such regulated splicing during cellular proliferation or differentiation.
Methyltransferases are enzymes fundamental to a wide range of normal biological activities that can become dysregulated during oncogenesis. For instance, the recent description of the ...methyltransferase-like (METTL) family of enzymes, has demonstrated the importance of the N
-adenosine-methyltransferase (m
A) modification in transcripts in the context of malignant transformation. Because of their importance, numerous METTL family members have been biochemically characterized to identify their cellular substrates, however some members such as METTL7B, recently renamed TMT1B and which is the subject of this review, remain enigmatic. First identified in the stacked Golgi, TMT1B is also localized to the endoplasmic reticulum as well as lipid droplets and has been reported as being upregulated in a wide range of cancer types including lung cancer, gliomas, and leukemia. Interestingly, despite evidence that TMT1B might act on protein substrates, it has also been shown to act on small molecule alkyl thiol substrates such as hydrogen sulfide, and its loss has been found to affect cellular proliferation and migration. Here we review the current evidence for TMT1B's activity, localization, and potential biological role in the context of both normal and cancerous cell types.
Chromosomal translocations involving KMT2A gene are one of the most common genetic alterations found in pediatric acute myeloid leukemias (AML) although the molecular mechanisms that initiate the ...disease remain incompletely defined. To elucidate these initiating events we have used a human model system of AML driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observe the development of a profound hypomethylation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem cell associated genes along with skewed expression in other genes such as S100A8/9 implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-seq data showed that there were relatively few leukemiaspecific changes and the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed the adenylate cyclase gene ADCY9 as an essential gene in KM3-AML, and suggest the potential for autocrine signalling through the chemokine receptor CCR1 and CCL23 ligand. Together, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.
Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is ...restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.
Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may ...play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.
The development of DNA sequencing more than 30 years ago has profoundly impacted biological research. In the last couple of years, remarkable technological innovations have emerged that allow the ...direct and cost-effective sequencing of complex samples at unprecedented scale and speed. These next-generation technologies make it feasible to sequence not only static genomes, but also entire transcriptomes expressed under different conditions. These and other powerful applications of next-generation sequencing are rapidly revolutionizing the way genomic studies are carried out. Below, we provide a snapshot of these exciting new approaches to understanding the properties and functions of genomes. Given that sequencing-based assays may increasingly supersede microarray-based assays, we also compare and contrast data obtained from these distinct approaches.
Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain–containing family of demethylases comprises 27 members that target ...mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.
•Jarid1b knockdown promotes enhanced HSC activity.
Despite the wealth of sequence data available from human, mouse and other genomes, our understanding of the mechanisms involved in regulating transcription and creating protein diversity is ...incomplete. Although effects such as alternative splicing have been extensively studied, other less well-characterized phenomena are now being recognized that increase the complexity of the transcribed portion of the genome. One mechanism, supported by a growing body of research, is the use of alternative promoters. In this article, we describe the consequences and significance of alternative promoter usage in human and mouse genomes, demonstrating that mammalian genes commonly employ multiple promoters to regulate and increase their transcriptional and translational potential.
The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) ...positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.
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