JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin.

Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. Here we show that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1alpha (HP1alpha), but not HP1beta, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1alpha at the same site. Tauhese results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia.

The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development.

The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.

Comparison of real-world treatment outcomes of femtosecond laser-assisted cataract surgery and phacoemulsification cataract surgery: A retrospective, observational study from an outpatient clinic in France.

PURPOSE: This study compared real-world safety and efficacy outcomes of cataract surgery performed with LenSx femtosecond laser-assisted cataract surgery or manual phacoemulsification cataract surgery procedures. METHODS: A retrospective observational study used data from anonymised electronic medical records to compare mean cumulative dissipated energy, the proportion of eyes reaching emmetropia, mean change in best-corrected distance visual acuity and the proportion of eyes with post-surgical complications, including corneal oedema and posterior capsule opacification. Results were adjusted for multiple comparisons for primary and secondary objectives. RESULTS: Data from 811 phacoemulsification cataract surgery and 496 femtosecond laser-assisted cataract surgery procedures were analysed. Mean cumulative dissipated energy was significantly lower for femtosecond laser-assisted cataract surgery (6.5 percent-seconds) than for phacoemulsification cataract surgery (14.3 percent-seconds; p < 0.0001) procedures. More femtosecond laser-assisted cataract surgery (81.2%) procedures achieved emmetropia (⩽ 0.5 dioptre) than did phacoemulsification cataract surgery (73.5%) procedures, although this difference was not statistically significant. Mean change in best-corrected distance visual acuity and the proportion of eyes with corneal oedema, posterior capsule opacification or other complications were not significantly different between cohorts when adjusted for multiple comparisons. CONCLUSIONS: In this single-centre, single-surgeon retrospective electronic medical record database study using divide and conquer technique, femtosecond laser-assisted cataract surgery was associated with significantly lower cumulative dissipated energy when compared to manual phacoemulsification cataract surgery. This supports the hypothesis that femtosecond laser-assisted cataract surgery involves less mechanical trauma, which might lead to more consistent refractive and safety outcomes than manual phacoemulsification cataract surgery, though such outcomes were found to be comparable in this study.

Three distinct patterns of histone H3Y41 phosphorylation mark active genes.

The JAK2 tyrosine kinase is a critical mediator of cytokine-induced signaling. It plays a role in the nucleus, where it regulates transcription by phosphorylating histone H3 at tyrosine 41 (H3Y41ph). We used chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq) to define the genome-wide pattern of H3Y41ph in human erythroid leukemia cells. Our results indicate that H3Y41ph is located at three distinct sites: (1) at a subset of active promoters, where it overlaps with H3K4me3, (2) at distal cis-regulatory elements, where it coincides with the binding of STAT5, and (3) throughout the transcribed regions of active, tissue-specific hematopoietic genes. Together, these data extend our understanding of this conserved and essential signaling pathway and provide insight into the mechanisms by which extracellular stimuli may lead to the coordinated regulation of transcription.

Genome-wide analysis of transcriptional reprogramming in mouse models of acute myeloid leukaemia.

Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML) using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer.

Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria.

We have developed a method for rapidly inactivating proteins with rapamycin-induced heterodimerization. Cells were stably transfected with siRNA-resistant, FKBP-tagged subunits of the adaptor protein (AP) complexes of clathrin-coated vesicles (CCVs), together with an FKBP and rapamycin-binding domain-containing construct with a mitochondrial targeting signal. Knocking down the endogenous subunit with siRNA, and then adding rapamycin, caused the APs to be rerouted to mitochondria within seconds. Rerouting AP-2 to mitochondria effectively abolished clathrin-mediated endocytosis of transferrin. In cells with rerouted AP-1, endocytosed cation-independent mannose 6-phosphate receptor (CIMPR) accumulated in a peripheral compartment, and isolated CCVs had reduced levels of CIMPR, but normal levels of the lysosomal hydrolase DNase II. Both observations support a role for AP-1 in retrograde trafficking. This type of approach, which we call a "knocksideways," should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days.

Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators.

Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1(+/-)::Gata2(+/-) compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.

From genes to cells to tissues--modelling the haematopoietic system.

Haematopoiesis (or blood formation) in general and haematopoietic stem cells more specifically represent some of the best studied mammalian developmental systems. Sophisticated purification protocols coupled with powerful biological assays permit functional analysis of highly purified cell populations both in vitro and in vivo. However, despite several decades of intensive research, the sheer complexity of the haematopoietic system means that many important questions remain unanswered or even unanswerable with current experimental tools. Scientists have therefore increasingly turned to modelling to tackle complexity at multiple levels ranging from networks of genes to the behaviour of cells and tissues. Early modelling attempts of gene regulatory networks have focused on core regulatory circuits but have more recently been extended to genome-wide datasets such as expression profiling and ChIP-sequencing data. Modelling of haematopoietic cells and tissues has provided insight into the importance of phenotypic heterogeneity for the differentiation of normal progenitor cells as well as a greater understanding of treatment response for particular pathologies such as chronic myeloid leukaemia. Here we will review recent progress in attempts to reconstruct segments of the haematopoietic system. A variety of modelling strategies will be covered from small-scale, protein-DNA or protein-protein interactions to large scale reconstructions. Also discussed will be examples of how stochastic modelling may be applied to multi cell systems such as those seen in normal and malignant haematopoiesis.