Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival.

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn(-/-) erythroblasts had attenuated ex vivo expansion, and late stage Lyn(-/-) erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn(-/-) erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKα/ß (glycogen synthase kinase α/ß) phosphorylation and diminished p27(Kip1) induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro- and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling.

Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling.

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lyn(up/up)) on erythropoiesis and Epo receptor signaling. Adult Lyn(up/up) mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn(+/up) mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn(+/up) erythroid cell lines and primary CD71(+) Lyn(up/up) erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated-binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn(+/up) cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects.

Crystal structures of the Lyn protein tyrosine kinase domain in its Apo- and inhibitor-bound state.

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.

Liar, a novel Lyn-binding nuclear/cytoplasmic shuttling protein that influences erythropoietin-induced differentiation.

Erythropoiesis is primarily controlled by erythropoietin (Epo), which stimulates proliferation, differentiation, and survival of erythroid precursors. We have previously shown that the tyrosine kinase Lyn is critical for transducing differentiation signals emanating from the activated Epo receptor. A yeast 2-hybrid screen for downstream effectors of Lyn identified a novel protein, Liar (Lyn-interacting ankyrin repeat), which forms a multiprotein complex with Lyn and HS1 in erythroid cells. Interestingly, 3 of the ankyrin repeats of Liar define a novel SH3 binding region for Lyn and HS1. Liar also contains functional nuclear localization and nuclear export sequences and shuttles rapidly between the nucleus and cytoplasm. Ectopic expression of Liar inhibited the differentiation of normal erythroid progenitors, as well as immortalized erythroid cells. Significantly, Liar affected Epo-activated signaling molecules including Erk2, STAT5, Akt, and Lyn. These results show that Liar is a novel Lyn-interacting molecule that plays an important role in regulating intracellular signaling events associated with erythroid terminal differentiation.

Erythroid defects in TRalpha-/- mice.

Thyroid hormone and its cognate receptor (TR) have been implicated in the production of red blood cells. Here, we show mice deficient for TRalpha have compromised fetal and adult erythropoiesis. Erythroid progenitor numbers were significantly reduced in TRalpha(-/-) fetal livers, and transit through the final stages of maturation was impeded. In addition, immortalized TRalpha(-/-) erythroblasts displayed increased apoptosis and reduced capacity for proliferation and differentiation. Adult TRalpha(-/-) mice had lower hematocrit levels, elevated glucocorticoid levels, and an altered stress erythropoiesis response to hemolytic anemia. Most TRalpha(-/-) animals contained markedly altered progenitor numbers in their spleens. Strikingly, 20% of TRalpha(-/-) mice failed to elicit a stress erythropoiesis response and recovered very poorly from hemolytic anemia. We conclude that an underlying erythroid defect exists in TRalpha(-/-) mice, demon-strating the importance of TRalpha to the erythroid compartment.

Hls5 regulated erythroid differentiation by modulating GATA-1 activity.

Hemopoietic lineage switch (Hls) 5 and 7 were originally isolated as genes up-regulated during an erythroid-to-myeloid lineage switch. We have shown previously that Hls7/Mlf1 imposes a monoblastoid phenotype on erythroleukemic cells. Here we show that Hls5 impedes erythroid maturation by restricting proliferation and inhibiting hemoglobin synthesis; however, Hls5 does not influence the morphology of erythroid cells. Under the influence of GATA-1, Hls5 relocates from cytoplasmic granules to the nucleus where it associates with both FOG-1 and GATA-1. In the nucleus, Hls5 is able to suppress GATA-1-mediated transactivation and reduce GATA-1 binding to DNA. We conclude that Hls5 and Hls7/Mlf1 act cooperatively to induce biochemical and phenotypic changes associated with erythroid/myeloid lineage switching.

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule.

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogeneous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.

Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells.

We have shown previously that the Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. The importance of Lyn to red cell maturation has been highlighted by Lyn-/- mice developing anemia. Here we show that Lyn interacts with C-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators C-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk). Lyn phosphorylated Cbp on several tyrosine residues, including Tyr314, which recruited Csk/Ctk to suppress Lyn kinase activity. Intriguingly, phosphorylated Tyr314 also bound suppressor of cytokine signaling 1 (SOCS1), another well characterized negative regulator of cell signaling, resulting in elevated ubiquitination, and degradation of Lyn. In Epo-responsive primary cells and cell lines, Lyn rapidly phosphorylated Cbp, suppressing Lyn kinase activity via Csk/Ctk within minutes of Epo stimulation; hours later, SOCS1 bound to Cbp and was involved in the ubiquitination and turnover of Lyn protein. Thus, a single phosphotyrosine residue on Cbp coordinates a two-phase process involving distinct negative regulatory pathways to inactivate, then degrade, Lyn.

Cross-regulation of JAK and Src kinases.

Members of the Janus kinase (JAK) family, JAK1, JAK2, JAK3 and Tyk2 are intimately involved in the signalling events initiated by cytokines activating cell surface receptors. They are responsible for phosphorylating these receptors, which create docking sites for downstream molecules such as the signal transducer and activator of transcription family members. In addition, cytokine receptors associate with members of the Src family kinase (SFK). JAKs and SFK work in concert to activate many of the signalling molecules, with both kinase families required for optimal transmission of intracellular signals. JAKs and SFK are also required for the activation and recruitment of negative regulators of cytokine signalling, e.g., protein tyrosine phosphatases (PTPs) and suppressors of cytokine signalling. Aberrant activity of the JAK-Src kinase duet can result in hemopoietic abnormalities including leukaemia. Additionally, the recent identification of a somatic JAK2 mutation as the cause of polycythema vera, further highlights the clinical importance of these molecules.

Outer membrane protein 25-a mitochondrial anchor and inhibitor of stress-activated protein kinase-3.

Stress-activated protein kinase-3 (SAPK3) is unique amongst the mitogen-activated protein kinase (MAPK) family with its C-terminal 5 amino acids directing interaction with the PDZ domain-containing substrates alpha1-Syntrophin and SAP90/PSD95. Here, we identify three additional PDZ domain-containing binding partners, Lin-7C, Scribble, and outer membrane protein 25 (OMP25). This latter protein is localised together with SAPK3 at the mitochondria but it is not a SAPK3 substrate. Instead, OMP25 inhibits SAPK3 activity towards PDZ domain-containing substrates such as alpha1-Syntrophin and substrates without PDZ domains such as the mitochondrial protein Sab. This is a new mechanism for the regulation of SAPK3 and suggests that its intracellular activity should not be solely assessed by its phosphorylation status.

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis.

In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn(-/-) mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn(-/-) erythroblasts, and the phenotype of Lyn(-/-) animals was remarkably similar to GATA-1(low) animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn(-/-) animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn(-/-) mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.

New insights into the regulation of erythroid cells.

The regulation of erythroid cells is complex and occurs at multiple levels. Erythroid precursors, once committed to this lineage, develop in association with specific macrophages within erythroblastic islands. While erythropoietin (Epo) is the principal regulator of erythroid progenitors, other cytokines and nuclear hormones also play an important role in the maturation of these cells. Signalling from the Epo-receptor activates several pathways, including the JAK/STAT, ras/raf/MAP kinase and PI3 kinase/Akt cascades to promote cell survival, proliferation and differentiation. Transcription factors such as GATA-1, EKLF and NF-E2 are crucial for progression along the erythroid maturation pathway; these, and a myriad of other transcription factors, must be expressed at the correct developmental stage for normal red blood cells to be formed.

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor.

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.

Differential regulation of SOCS genes in normal and transformed erythroid cells.

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells - erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1-/- mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Enforced expression of HOX11 is associated with an immature phenotype in J2E erythroid cells.

The HOX11 gene encodes a homeodomain transcription factor that is essential for spleen development during embryogenesis. HOX11 is also leukaemogenic, both through its clinical association with childhood T-cell acute lymphoblastic leukaemia, and its ability to immortalize other haematopoietic cell lineages experimentally. To examine the pathological role of HOX11 in tumorigenesis, we constitutively expressed HOX11 cDNA in J2E murine erythroleukaemic cells, which are capable of terminal differentiation. Enforced HOX11 expression was found to induce a profound alteration in J2E cellular morphology and differentiation status. Our analyses revealed that HOX11 produced clones with a preponderance of less differentiated cells that were highly adherent to plastic. Morphologically, the cells overexpressing HOX11 were larger and had decreased globin levels, as well as a reduction in haemoglobin synthesis in response to erythropoietin (EPO). Immunocytochemical analysis confirmed the immature erythroid phenotype imposed by HOX11, with clones transfected with HOX11 demonstrating expression of the c-Kit stem cell marker, while retaining EPO receptor expression. Taken together, these results show that HOX11 alters erythroid differentiation, favouring a less mature progenitor-like stage. This supports the notion that disrupted haematopoietic cell differentiation is responsible for pre-leukaemic immortalization by the HOX11 oncoprotein.

MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.