Adam C. Wilkinson

Rfam: updates to the RNA families database.

Rfam is a collection of RNA sequence families, represented by multiple sequence alignments and covariance models (CMs). The primary aim of Rfam is to annotate new members of known RNA families on nucleotide sequences, particularly complete genomes, using sensitive BLAST filters in combination with CMs. A minority of families with a very broad taxonomic range (e.g. tRNA and rRNA) provide the majority of the sequence annotations, whilst the majority of Rfam families (e.g. snoRNAs and miRNAs) have a limited taxonomic range and provide a limited number of annotations. Recent improvements to the website, methodologies and data used by Rfam are discussed. Rfam is freely available on the Web at http://rfam.sanger.ac.uk/and http://rfam.janelia.org/.

Decoding the regulatory network of early blood development from single-cell gene expression measurements

Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

Summary The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.

Clonal dynamics reveal two distinct populations of basal cells in slow turnover airway epithelium

Summary Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.

CODEX: a next-generation sequencing experiment database for the haematopoietic and embryonic stem cell communities

CODEX (http://codex.stemcells.cam.ac.uk/) is a user-friendly database for the direct access and interrogation of publicly available next-generation sequencing (NGS) data, specifically aimed at experimental biologists. In an era of multi-centre genomic dataset generation, CODEX provides a single database where these samples are collected, uniformly processed and vetted. The main drive of CODEX is to provide the wider scientific community with instant access to high-quality NGS data, which, irrespective of the publishing laboratory, is directly comparable. CODEX allows users to immediately visualize or download processed datasets, or compare user-generated data against the databases cumulative knowledge-base. CODEX contains four types of NGS experiments: transcription factor chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), histone modification ChIP-Seq, DNase-Seq and RNA-Seq. These are largely encompassed within two specialized repositories, HAEMCODE and ESCODE, which are focused on haematopoiesis and embryonic stem cell samples, respectively. To date, CODEX contains over 1000 samples, including 221 unique TFs and 93 unique cell types. CODEX therefore provides one of the most complete resources of publicly available NGS data for the direct interrogation of transcriptional programmes that regulate cellular identity and fate in the context of mammalian development, homeostasis and disease.

Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation

Hematopoietic stem cell maintenance requires the amino acid valine. How to maintain hematopoietic stem cells Hematopoiesis provides the body with a continuous supply of blood cells (see the Perspective by Sommerkamp and Trumpp). Taya et al. report that amino acid content is important for hematopoietic stem cell (HSC) maintenance in vitro and in vivo. Dietary valine restriction seems to “empty” the mouse bone marrow niche. Ito et al. used single-cell approaches and cell transplantation to identify a subset of HSCs at the top of the HSC hierarchy. Self-renewal relied on the induction of mitophagy, a quality-control process linked to a cells metabolic state. Both studies may be helpful in improving clinical bone marrow transplantation. Science, this issue p. 1103, p. 1152; see also p. 1156 A specialized bone marrow microenvironment (niche) regulates hematopoietic stem cell (HSC) self-renewal and commitment. For successful donor-HSC engraftment, the niche must be emptied via myeloablative irradiation or chemotherapy. However, myeloablation can cause severe complications and even mortality. Here we report that the essential amino acid valine is indispensable for the proliferation and maintenance of HSCs. Both mouse and human HSCs failed to proliferate when cultured in valine-depleted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within 1 week. Furthermore, dietary valine restriction emptied the mouse bone marrow niche and afforded donor-HSC engraftment without chemoirradiative myeloablation. These findings indicate a critical role for valine in HSC maintenance and suggest that dietary valine restriction may reduce iatrogenic complications in HSC transplantation.

Transcriptional regulation of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell population found in the bone marrow of adult mammals and are responsible for maintaining the entire haematopoietic system. Definitive HSCs are produced from mesoderm during embryonic development, from embryonic day 10 in the mouse. HSCs seed the foetal liver before migrating to the bone marrow around the time of birth. In the adult, HSCs are largely quiescent but have the ability to divide to self-renew and expand, or to proliferate and differentiate into any mature haematopoietic cell type. Both the specification of HSCs during development and their cellular choices once formed are tightly controlled at the level of transcription. Numerous transcriptional regulators of HSC specification, expansion, homeostasis and differentiation have been identified, primarily from analysis of mouse gene knockout experiments and transplantation assays. These include transcription factors, epigenetic modifiers and signalling pathway effectors. This chapter reviews the current knowledge of these HSC transcriptional regulators, predominantly focusing on the transcriptional regulation of mouse HSCs, although transcriptional regulation of human HSCs is also mentioned where relevant. Due to the breadth and maturity of this field, we have prioritised recently identified examples of HSC transcriptional regulators. We go on to highlight additional layers of control that regulate expression and activity of HSC transcriptional regulators and discuss how chromosomal translocations that result in fusion proteins of these HSC transcriptional regulators commonly drive leukaemias through transcriptional dysregulation.

NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor.

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD+‐dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD+ as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature‐sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD+‐dependent DNA ligases. Proteins 2003;51:321–326.

Integrated genome-scale analysis of the transcriptional regulatory landscape in a blood stem/progenitor cell model

Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor-dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology.

Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification

Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood. Transcription activator-like effectors (TALEs) are a novel class of genetic tool based on the modular DNA-binding domains of Xanthomonas TAL proteins, which enable DNA sequence-specific targeting and the manipulation of endogenous gene expression. Here, we report TALEs engineered to target the PU.1-14kb and Scl+40kb transcriptional enhancers as efficient new tools to perturb the expression of these key haematopoietic TFs. We confirmed the efficiency of these TALEs at the single-cell level using high-throughput RT-qPCR, which also allowed us to assess the consequences of both PU.1 activation and repression on wider TF networks during developmental haematopoiesis. Combined with comprehensive cellular assays, these experiments uncovered novel roles for PU.1 during early haematopoietic specification. Finally, transgenic mouse studies confirmed that the PU.1-14kb element is active at sites of definitive haematopoiesis in vivo and PU.1 is detectable in haemogenic endothelium and early committing blood cells. We therefore establish TALEs as powerful new tools to study the functionality of transcriptional networks that control developmental processes such as early haematopoiesis.