On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate.

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes.

Reactivation of a developmentally silenced embryonic globin gene.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin.

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal.

Phenotypic and molecular characterization of a serum-free miniature erythroid differentiation system suitable for high-throughput screening and single-cell assays.

In vitro erythroid differentiation systems are used to study the mechanisms underlying normal and abnormal erythropoiesis and to test the effects of various extracellular factors on erythropoiesis. The use of serum or conditioned medium in liquid cultures and the seeding of cultures with heterogeneous peripheral blood mononuclear cells confound the reproducibility of these systems. Newer erythroid differentiation culture systems have overcome some of these limitations by using a fully defined, serum-free medium and initiating cultures using purified CD34 cells.

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia.

β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity.

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment.

The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats.

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG) ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation.

High-resolution analysis of cis-acting regulatory networks at the α-globin locus.

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster.

Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown.

Nprl3 is required for normal development of the cardiovascular system.

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway.

Intragenic enhancers act as alternative promoters.

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure.

Polycomb eviction as a new distant enhancer function.

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island.

Generation of bivalent chromatin domains during cell fate decisions.

Background: In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined.

Results: Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus.

Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts.

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained.

Human induced pluripotent stem cells are capable of B-cell lymphopoiesis.

Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells.

Lineage-specific combinatorial action of enhancers regulates mouse erythroid Gata1 expression.

Precise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS-3.5, can direct both erythroid and megakaryocytic expression.

Chromosome looping at the human alpha-globin locus is mediated via the major upstream regulatory element (HS -40).

Previous studies in the mouse have shown that high levels of alpha-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human alpha cluster and their interactions with the alpha-globin promoter. All of these elements interact with the alpha-globin gene in an erythroid-specific manner.

The mouse Runx1 +23 hematopoietic stem cell enhancer confers hematopoietic specificity to both Runx1 promoters.

The transcription factor Runx1 plays a pivotal role in hematopoietic stem cell (HSC) emergence, and studies into its transcriptional regulation should give insight into the critical steps of HSC specification. Recently, we identified the Runx1 +23 enhancer that targets reporter gene expression to the first emerging HSCs of the mouse embryo when linked to the heterologous hsp68 promoter. Endogenous Runx1 is transcribed from 2 alternative promoters, P1 and P2.