Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad-a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models.

Epstein-Barr virus (EBV) infects nearly all humans and usually is asymptomatic, or in the case of adolescents and young adults, it can result in infectious mononucleosis. EBV-infected B cells are controlled primarily by NK cells, iNKT cells, CD4 T cells, and CD8 T cells. While mutations in proteins important for B cell function can affect EBV infection of these cells, these mutations do not result in severe EBV infection. Some genetic disorders affecting T and NK cell function result in failure to control EBV infection, but do not result in increased susceptibility to other virus infections. These include mutations in SH2D1A, BIRC4, ITK, CD27, MAGT1, CORO1A, and LRBA. Since EBV is the only virus that induces proliferation of B cells, the study of these diseases has helped to identify proteins critical for interactions of T and/or NK cells with B cells. Mutations in three genes associated with hemophagocytic lymphohistocytosis, PRF1, STXBP2, and UNC13D, can also predispose to severe chronic active EBV disease. Severe EBV infection can be associated with immunodeficiencies that also predispose to other viral infections and in some cases other bacterial and fungal infections. These include diseases due to mutations in PIK3CD, PIK3R1, CTPS1, STK4, GATA2, MCM4, FCGR3A, CARD11, ATM, and WAS. In addition, patients with severe combined immunodeficiency, which can be due to mutations in a number of different genes, are at high risk for various infections as well as EBV B cell lymphomas. Identification of proteins important for control of EBV may help to identify new targets for immunosuppressive therapies.

Polycomb repressive complex 2 (PRC2) plays crucial roles in transcriptional regulation and stem cell development. However, the context-specific functions associated with alternative subunits remain largely unexplored. Here we show that the related enzymatic subunits EZH1 and EZH2 undergo an expression switch during blood cell development. An erythroid-specific enhancer mediates transcriptional activation of EZH1, and a switch from GATA2 to GATA1 controls the developmental EZH1/2 switch by differential association with EZH1 enhancers. We further examine the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and reveal the existence of an EZH1-SUZ12 subcomplex lacking EED. EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin, and positively regulate gene expression. Loss of EZH2 expression leads to repositioning of EZH1 to EZH2 targets. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical functions during blood stem cell specification.

Epigenetic histone modifications play critical roles in the control of self-renewal and differentiation of hematopoietic stem cells (HSCs). Mysm1 is a recently identified histone H2A deubiquitinase with essential and intrinsic roles for maintaining functional HSCs. In this study, in addition to confirming this function of Mysm1, by using Mysm1-deficient (Mysm1(-/-)) mice, we provide more evidence for how Mysm1 controls HSC homeostasis. Mysm1 deletion drives HSCs from quiescence into rapid cycling and increases their apoptotic rate, resulting in an exhaustion of the stem cell pool, which leads to an impaired self-renewal and lineage reconstituting abilities in the Mysm1-deficient mice. Our study identified Gfi1 as one of the candidate genes responsible for the HSC defect in Mysm1-deficient mice. Mechanistic studies revealed that Mysm1 modulates histone modifications and directs the recruitment of key transcriptional factors such as Gata2 and Runx1 to the Gfi1 locus in HSCs. We found that Mysm1 directly associates with the Gfi1 enhancer element and promotes its transcription through Gata2 and Runx1 transactivation. Thus, our study not only elaborates on the initial reports of Mysm1 association with HSC homeostasis but also delineates a possible epigenetic mechanism through which Mysm1 carries out this function in the HSCs.

Autoimmunity and immune dysregulation may lead to cytopenia and represent key features of many primary immunodeficiencies (PIDs). Especially when cytopenia is the initial symptom of a PID, the order and depth of diagnostic steps have to be performed in accordance with both an immunologic and a hematologic approach and will help exclude disorders such as systemic lupus erythematosus, common variable immunodeficiency, and autoimmune lymphoproliferative syndromes, hemophagocytic disorders, lymphoproliferative diseases, and novel differential diagnoses such as MonoMac syndrome (GATA2 deficiency), CD27 deficiency, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, activated PI3KD syndrome (APDS), X-linked immunodeficiency with magnesium defect (MAGT1 deficiency), and others. Immunosuppressive treatment often needs to be initiated urgently, which impedes further relevant immunologic laboratory analyses aimed at defining the underlying PID. Awareness of potentially involved disease spectra ranging from hematologic to rheumatologic and immunologic disorders is crucial for identifying a certain proportion of PID phenotypes and genotypes among descriptive diagnoses such as autoimmune hemolytic anemia, chronic immune thrombocytopenia, Evans syndrome, severe aplastic anemia/refractory cytopenia, and others. A synopsis of pathomechanisms, novel differential diagnoses, and advances in treatment options for cytopenias in PID is provided to facilitate multidisciplinary management and to bridge different approaches.

The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif. Putative EVI1 target genes were identified by using an EVI1-(1-250)-VP16 fusion protein that acts as a transcriptional activator with the binding specificity of EVI1. Sixteen genes induced in NIH 3T3 cells by wild type EVI1-VP16 but not by mutant forms were identified. Sequence analysis revealed evolutionarily conserved GACAAGATA-like motifs within 10 kb of their transcription start sites, and by chromatin immunoprecipitation in fibroblasts, we showed occupancy of many of these sites by EVI1-VP16. To assess whether native EVI1 binds to these sites in EVI1-transformed myeloid cells, we performed chromatin immunoprecipitation in 32Dcl3 and NFS58 cells, using anti-EVI1 antisera, and we showed that the majority of these sites is bound by wild type EVI1. These putative target genes include Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik). In this study we demonstrated for the first time that the N-terminal DNA binding domain of EVI1 has the capacity to bind to endogenous genes. We hypothesized that these genes play a critical role in EVI1-induced transformation.

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells, Lin(-)c-Kit+ cells, and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid, B-lymphoid, and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3, MPO, Neutrophil elastase, Cathepsin G, and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1, Notch1, LEF-1, Mcl-1, and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development.

Although most patients with myelofibrosis (MF) derive benefit from ruxolitinib, some are refractory, have a suboptimal response, or quickly lose their response. To identify genes that may predict response to ruxolitinib, we performed targeted next-generation sequencing (NGS) of a panel of 28 genes recurrently mutated in hematologic malignancies in a cohort of patients with MF who were treated with ruxolitinib in a phase 1/2 study. We also tested for CALR deletions by standard polymerase chain reaction methods. Ninety-eight percent of patients had a mutation in ≥1 gene. Seventy-nine (82.1%) patients had the JAK2(V617F) mutation, 9 (9.5%) had CALR mutations (7 type 1, 2 type 2), 3 (3.1%) had MPL mutations, and 4 (4.2%) were negative for all 3. ASXL1/JAK2 and TET2/JAK2 were the most frequently comutated genes. Mutations in NRAS, KRAS, PTPN11, GATA2, TP53, and RUNX1 were found in <5% of patients. Spleen response (≥50% reduction in palpable spleen size) was inversely correlated with the number of mutations; patients with ≤2 mutations had ninefold higher odds of a spleen response than those with ≥3 mutations (odds ratio = 9.37; 95% confidence interval, 1.86-47.2). Patients with ≥3 mutations also had a shorter time to treatment discontinuation and shorter overall survival than those with fewer mutations. In multivariable analysis, only number of mutations and spleen response remained associated with time to treatment discontinuation. Patients with ≥3 mutations had the worst outcomes, suggesting that multigene profiling may be useful for therapeutic planning for MF.

Germ-line GATA2 gene mutations, leading to haploinsufficiency, have been identified in patients with familial myelodysplastic syndrome/acute myeloid leukemia, monocytopenia and mycobacterial infections, Emberger syndrome, and dendritic cell, monocyte, B-, and NK-cell deficiency. GATA2 mutations have also been reported in a minority of patients with congenital neutropenia and aplastic anemia (AA). The bone marrow (BM) from patients with GATA2 deficiency is typically hypocellular, with varying degrees of dysplasia. Distinguishing GATA2 patients from those with AA is critical for selecting appropriate therapy. We compared the BM flow cytometric, morphologic, and cytogenetic features of 28 GATA2 patients with those of 32 patients being evaluated for idiopathic AA. The marrow of GATA2 patients had severely reduced monocytes, B cells, and NK cells; absent hematogones; and inverted CD4:CD8 ratios. Atypical megakaryocytes and abnormal cytogenetics were more common in GATA2 marrows. CD34(+) cells were comparably reduced in GATA2 and AA. Using these criteria, we prospectively identified 4 of 32 patients with suspected AA who had features suspicious for GATA2 mutations, later confirmed by DNA sequencing. Our results show that routine BM flow cytometry, morphology, and cytogenetics in patients who present with cytopenia(s) can identify patients for whom GATA2 sequencing is indicated.

The cis- and trans-acting factors that are critical for placenta-specific expression of the human syncytin gene are unknown. We identified a 146-base pair (bp) region of the 5'-flanking region of the human syncytin gene from nt-294 to -148 that is essential for basal gene expression in human BeWo and JEG3 choriocarcinoma cell lines but not in hepatoblastoma and kidney cell lines. Ligation of the 146-bp fragment to a SV40 promoter or a human beta-globin minimal promoter markedly enhanced promoter activity in the placenta cells but not in the liver and kidney cells. DNase I footprint assays indicated that nuclear extracts from BeWo cells but not HepG2 cells protected four regions (FP1-FP4) of the 146-bp fragment. Site-directed mutagenesis of an SP1-binding site in FP3 and a GATA-binding site in FP4 significantly repressed promoter activity in the placenta cells. Overexpression of SP1 (Sp1 transcription factor) and GATA2 (GATA binding protein 2) and GATA3 induced syncytin promoter activity but had little or no effect on the activities of syncytin promoter fragments containing mutations in the SP1- and GATA-binding sites. GATA2 and -3 mRNA levels increased markedly during spontaneous in vitro differentiation of human cytotrophoblast cells when the cytotrophoblast cells fused to form a syncytium. These findings strongly suggest that the 146-bp region of the 5'-flanking region (nt-294/-148) of the human syncytin gene acts as a placenta-specific enhancer. Binding of SP1 and GATA family members to this enhancer is critical for cell-specific expression of the syncytin gene.

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.

Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo. Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis. We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development. The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene. Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence. Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity. We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3' to the Gata2 structural gene. Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.

Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. The cofactor FOG1 interacts with transcription factors GATA1 and GATA2 to control erythroid and megakaryocyte (MK) differentiation. In contrast, FOG1 antagonizes the ability of GATA factors to promote mast cell (MC) development. Normal FOG1 function in late-stage erythroid cells and MK requires interaction with the chromatin remodeling complex NuRD. Here, we report that mice in which the FOG1/NuRD interaction is disrupted (Fog(ki/ki)) produce MK-erythroid progenitors that give rise to significantly fewer and less mature MK and erythroid colonies in vitro while retaining multilineage capacity, capable of generating MCs and other myeloid lineage cells. Gene expression profiling of Fog(ki/ki) MK-erythroid progenitors revealed inappropriate expression of several MC-specific genes. Strikingly, aberrant MC gene expression persisted in mature Fog(ki/ki) MK and erythroid progeny. Using a GATA1-dependent committed erythroid cell line, select MC genes were found to be occupied by NuRD, suggesting a direct mechanism of repression. Together, these observations suggest that a simple heritable silencing mechanism is insufficient to permanently repress MC genes. Instead, the continuous presence of GATA1, FOG1, and NuRD is required to maintain lineage fidelity throughout MK-erythroid ontogeny.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.

Endoglin (Eng), an accessory receptor for the transforming growth factor β (TGF-β) superfamily, is required for proper hemangioblast and primitive hematopoietic development. However the mechanism by which endoglin functions at this early developmental stage is currently unknown. Transcriptional analyses of differentiating eng(-/-) and eng(+/+) ES cells revealed that lack of endoglin leads to profound reductions in the levels of key hematopoietic regulators, including Scl, Lmo2, and Gata2. We also detected lower levels of phosphorylated Smad1 (pSmad1), a downstream target signaling molecule associated with the TGF-β pathway. Using doxycycline-inducible ES cell lines, we interrogated the TGF-β signaling pathway by expressing activated forms of ALK-1 and ALK-5, type I receptors for TGF-β. Our results indicate that ALK-1 signaling promotes hemangioblast development and hematopoiesis, as evidenced by colony assays, gene expression and FACS analyses, whereas signaling by ALK-5 leads to the opposite effect, inhibition of hemangioblast and hematopoietic development. In Eng(-/-) ES cells, ALK-1 rescued both the defective hemangioblast development, and primitive erythropoiesis, indicating that ALK-1 signaling can compensate for the absence of endoglin. We propose that endoglin regulates primitive hematopoiesis by modulating the activity of the Smad1/5 signaling pathway in early stages of development.

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Forced expression of the transcription factor HoxB4 has been shown to enhance the self-renewal capacity of mouse bone marrow hematopoietic stem cells (HSCs) and confer a long-term repopulating capacity to yolk sac and embryonic stem (ES) cell-derived hematopoietic precursors. The fact that ES cell-derived precursors do not repopulate bone marrow without HoxB4 underscores an important role for HoxB4 in the maturation of ES-derived hematopoietic precursors into long-term repopulating HSCs. However, the precise molecular mechanism underlying this process is barely understood. In this study, we performed a genome-wide analysis of HoxB4 using ES cell-derived hematopoietic stem/progenitor cells. The results revealed many of the genes essential for HSC development to be direct targets of HoxB4, such as Runx1, Scl/Tal1, Gata2, and Gfi1. The expression profiling also showed that HoxB4 indirectly affects the expression of several important genes, such as Lmo2, Erg, Meis1, Pbx1, Nov, AhR, and Hemgn. HoxB4 tended to activate the transcription, but the down-regulation of a significant portion of direct targets suggested its function to be context-dependent. These findings indicate that HoxB4 reprograms a set of key regulator genes to facilitate the maturation of developing HSCs into repopulating cells. Our list of HoxB4 targets also provides novel candidate regulators for HSCs.

BACKGROUND

The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM).

METHODS

LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry.

RESULTS

The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues.

CONCLUSIONS

The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

Osteoclasts are bone-resorbing cells essential for skeletal development, homeostasis, and regeneration. They derive from hematopoietic progenitors in the monocyte/macrophage lineage and differentiate in response to RANKL. However, the precise nature of osteoclast progenitors is a longstanding and important question. Using inducible peroxisome proliferator-activated receptor γ (PPARγ)-tTA TRE-GFP (green fluorescent protein) reporter mice, we show that osteoclast progenitors reside specifically in the PPARγ-expressing hematopoietic bone marrow population and identify the quiescent PPARγ(+) cells as osteoclast progenitors. Importantly, two PPARγ-tTA TRE-Cre-controlled genetic models provide compelling functional evidence. First, Notch activation in PPARγ(+) cells causes high bone mass due to impaired osteoclast precursor proliferation. Second, selective ablation of PPARγ(+) cells by diphtheria toxin also causes high bone mass due to decreased osteoclast numbers. Furthermore, PPARγ(+) cells respond to both pathological and pharmacological resorption-enhancing stimuli. Mechanistically, PPARγ promotes osteoclast progenitors by activating GATA2 transcription. These findings not only identify the long-sought-after osteoclast progenitors but also establish unprecedented tools for their visualization, isolation, characterization, and genetic manipulation.

Canonical Wnt ligands are secreted by several cell types in the adipose tissue. We examined if mature adipocytes can also be target cells and found that canonical Wnt activation by Wnt3a induced a marked dedifferentiation of both 3T3-L1 and human adipocytes. Typical adipogenic markers were reduced while undifferentiated cell markers like Pref-1/Dlk1, Wnt10b, and Gata2 were increased. The cells also became insulin-resistant with impaired upstream insulin signaling and reduced glucose uptake. Wnt3a stabilized beta-catenin in the absence of the LRP6 receptor and with maintained axin and Dickkopf-1 protein expression. PPARgamma was repressed and PPARgamma ligands could not restore the adipogenic markers or reduce the beta-catenin levels. The dedifferentiated adipocytes expressed the myofibroblast marker alpha-smooth muscle actin and were also susceptible to osteogenic transdifferentiation. These results identify a novel pathway in mature adipose cells that is critical for maintaining the normal adipocyte phenotype and insulin sensitivity.

As with all human herpesviruses, human cytomegalovirus (HCMV) persists for the lifetime of the host by establishing a latent infection, which is broken by periodic reactivation events. One site of HCMV latency is in the progenitor cells of the myeloid lineage such as CD34+ cells and their CD14+ derivatives. The development of experimental techniques to isolate and culture these primary cells in vitro is enabling detailed analysis of the events that occur during virus latency and reactivation. Ex vivo differentiation of latently infected primary myeloid cells to dendritic cells and macrophages results in the reactivation of latent virus and provides model systems in which to analyse the viral and cellular functions involved in latent carriage and reactivation. Such analyses have shown that, in contrast to primary lytic infection or reactivation which is characterised by a regulated cascade of expression of all viral genes, latent infection is associated with a much more restricted viral transcription programme with expression of only a small number of viral genes. Additionally, concomitant changes in the expression of cellular miRNAs and cellular proteins occur, and this includes changes in the expression of a number of secreted cellular proteins and intracellular anti-apoptotic proteins, which all have profound effects on the latently infected cells. In this review, we concentrate on the effects of one of the latency-associated viral proteins, LAcmvIL-10, and describe how it causes a decrease in the cellular miRNA, hsa-miR-92a, and a concomitant upregulation of the GATA2 myeloid transcription factor, which, in turn, drives the expression of cellular IL-10. Taken together, we argue that HCMV latency, rather than a period of viral quiescence, is associated with the virally driven manipulation of host cell functions, perhaps every bit as complex as lytic infection. A full understanding of these changes in cellular and viral gene expression during latent infection could have far-reaching implications for therapeutic intervention.

Different mechanisms for CBFβ-MYH11 function in acute myeloid leukemia with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFβ-MYH11-binding site analysis and quantitative interaction proteomics, we found that CBFβ-MYH11 localizes to RUNX1 occupied promoters, where it interacts with TAL1, FLI1 and TBP-associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the coregulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knockdown, a small subset of the CBFβ-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFβ-MYH11 target genes, including genes implicated in hematopoietic stem cell self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knockdown. Together these results suggest an essential role for CBFβ-MYH11 in regulating the expression of genes involved in maintaining a stem cell phenotype.

A network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. However, choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of ∼2,000 TFs. Here, we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1, Mef2c, Esx1, Foxa1, Hnf4a, Gata2, Gata3, Myc, Elf5, Irf2, Elf1, Sfpi1, Ets1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1, Sox2, or Ascl1-can direct efficient, specific, and rapid differentiation into myocytes, hepatocytes, blood cells, and neurons. Furthermore, transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation, and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates.