PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate.

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.

PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K.

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

L-plastin associated syndrome of immune deficiency and hematologic cytopenia.

BACKGROUND: LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects. OBJECTIVE: This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. METHODS: We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. RESULTS: A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation. CONCLUSIONS: We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies.

BACKGROUND: TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. OBJECTIVE: We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. METHODS: Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. RESULTS: Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating B cells but overall normal humoral immune responses. CONCLUSION: Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

ERG promotes T-acute lymphoblastic leukemia and is transcriptionally regulated in leukemic cells by a stem cell enhancer.

The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia.

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F-positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2(V617F) mice develop reduced numbers of lineage(-)Sca-1(+)c-Kit(+) cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2(V617F) mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Myeloid transforming protein Evi1 interacts with methyl-CpG binding domain protein 3 and inhibits in vitro histone deacetylation by Mbd3/Mi-2/NuRD.

The ecotropic viral integration site 1 ( Evi1) gene encodes a putative transcription regulator, which is aberrantly expressed in acute myeloid leukemias (AML) with chromosomal abnormalities involving the 3q26 locus. Repression and activation of transcriptional control have been reported, but it is currently unclear how Evi1 may evoke these opposing effects. Using a yeast two-hybrid screen, we identified a novel binding partner of Evi1, i.e., methyl binding domain 3b (Mbd3b) protein, a member of the Mi-2/NuRD histone deacetylase complex. Applying in vitro and in vivo assays, we found that Evi1 interacts with Mbd3b but not with other MBD family members Mbd1, -2, and -4 or MeCP2. We show that interaction of Evi1 with Mbd3 requires 40 amino acids that are adjacent and downstream of the methyl binding domain (MBD). We further demonstrate that the first three zinc fingers of Evi1 are needed for Mbd3 interaction. Evi1 acts as a transcriptional repressor when recruited to an active promoter, yet when present in the Mi-2/NuRD complex through Mbd3 interaction, it inhibits the histone deacetylation function of this multiprotein structure. Our data may in part explain how Evi1 could act as a repressor as well as an activator of transcription.

A novel interaction between the proto-oncogene Evi1 and histone methyltransferases, SUV39H1 and G9a.

The transcription factor ecotropic viral integration site 1 (Evi1) is associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in patients due to chromosomal aberration of chromosome 3. Here we show that Evi1 interacts with the histone methyltransferase SUV39H1. The interaction requires the N-terminal part of Evi1 and the H3-specific histone methyltransferase domain, SET, of SUV39H1 without Evi1 having an inhibitory effect on SUV39H1 methyltransferase activity. Presence of SUV39H1 enhances Evi1 transcriptional repression in a dose dependent manner. In addition, Evi1 also interacts with another histone methyltransferase, G9a, but not with SET9. Our data establish an epigenetic role of Evi1 in cell transformation by recruiting higher order chromatin remodeling complexes.

SETDB1 prevents TET2-dependent activation of IAP retroelements in naïve embryonic stem cells.

BACKGROUND: Endogenous retroviruses (ERVs), which are responsible for 10% of spontaneous mouse mutations, are kept under control via several epigenetic mechanisms. The H3K9 histone methyltransferase SETDB1 is essential for ERV repression in embryonic stem cells (ESCs), with DNA methylation also playing an important role. It has been suggested that SETDB1 protects ERVs from TET-dependent DNA demethylation, but the relevance of this mechanism for ERV expression remains unclear. Moreover, previous studies have been performed in primed ESCs, which are not epigenetically or transcriptionally representative of preimplantation embryos. RESULTS: We use naïve ESCs to investigate the role of SETDB1 in ERV regulation and its relationship with TET-mediated DNA demethylation. Naïve ESCs show an increased dependency on SETDB1 for ERV silencing when compared to primed ESCs, including at the highly mutagenic intracisternal A particles (IAPs). We find that in the absence of SETDB1, TET2 activates IAP elements in a catalytic-dependent manner. Surprisingly, TET2 does not drive changes in DNA methylation levels at IAPs, suggesting that it regulates these retrotransposons indirectly. Instead, SETDB1 depletion leads to a TET2-dependent loss of H4R3me2s, which is indispensable for IAP silencing during epigenetic reprogramming. CONCLUSIONS: Our results demonstrate a novel and unexpected role for SETDB1 in protecting IAPs from TET2-dependent histone arginine demethylation.

Compensation between CSF1R+ macrophages and Foxp3+ Treg cells drives resistance to tumor immunotherapy.

Redundancy and compensation provide robustness to biological systems but may contribute to therapy resistance. Both tumor-associated macrophages (TAMs) and Foxp3+ regulatory T (Treg) cells promote tumor progression by limiting antitumor immunity. Here we show that genetic ablation of CSF1 in colorectal cancer cells reduces the influx of immunosuppressive CSF1R+ TAMs within tumors. This reduction in CSF1-dependent TAMs resulted in increased CD8+ T cell attack on tumors, but its effect on tumor growth was limited by a compensatory increase in Foxp3+ Treg cells. Similarly, disruption of Treg cell activity through their experimental ablation produced moderate effects on tumor growth and was associated with elevated numbers of CSF1R+ TAMs. Importantly, codepletion of CSF1R+ TAMs and Foxp3+ Treg cells resulted in an increased influx of CD8+ T cells, augmentation of their function, and a synergistic reduction in tumor growth. Further, inhibition of Treg cell activity either through systemic pharmacological blockade of PI3Kδ, or its genetic inactivation within Foxp3+ Treg cells, sensitized previously unresponsive solid tumors to CSF1R+ TAM depletion and enhanced the effect of CSF1R blockade. These findings identify CSF1R+ TAMs and PI3Kδ-driven Foxp3+ Treg cells as the dominant compensatory cellular components of the immunosuppressive tumor microenvironment, with implications for the design of combinatorial immunotherapies.

An endosiRNA-Based Repression Mechanism Counteracts Transposon Activation during Global DNA Demethylation in Embryonic Stem Cells.

Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.

Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia.

ETV6 (ets translocation variant gene 6) TEL (translocation ets leukemia), encoding a transcriptional repressor, is involved in various translocations associated with human malignancies. Strikingly, the nonrearranged ETV6 allele is often deleted or inactivated in cells harboring these translocations. Although ETV6 translocations are infrequent in acute myeloid leukemia (AML), mutations or deregulated expression of ETV6 may contribute to leukemogenesis. To investigate the involvement of ETV6 in AML, we analysed 300 newly diagnosed patients for mutations in the coding region of the gene. Furthermore, we studied protein expression in 77 patients using two ETV6-specific antibodies. Five somatic heterozygous mutations were detected, which affected either the homodimerization- or the DNA-binding domain of ETV6. The proteins translated from the cDNAs of these mutants were unable to repress transcription and showed dominant-negative effects. In addition, we demonstrate that one-third of AML patients have deficient ETV6 protein expression, which is not related to ETV6 mRNA expression levels. In conclusion, we demonstrate that ETV6 abnormalities are not restricted to translocations and occur more frequently in AML than previously thought. Additional comprehensive studies are required to define the clinical consequence of ETV6 loss of function in AML.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

Impaired in vitro erythropoiesis following deletion of the Scl (Tal1) +40 enhancer is largely compensated for in vivo despite a significant reduction in expression.

The Scl (Tal1) gene encodes a helix-loop-helix transcription factor essential for hematopoietic stem cell and erythroid development. The Scl +40 enhancer is situated downstream of Map17, the 3' flanking gene of Scl, and is active in transgenic mice during primitive and definitive erythropoiesis. To analyze the in vivo function of the Scl +40 enhancer within the Scl/Map17 transcriptional domain, we deleted this element in the germ line. Scl(Δ40/Δ40) mice were viable with reduced numbers of erythroid CFU in both bone marrow and spleen yet displayed a normal response to stress hematopoiesis. Analysis of Scl(Δ40/Δ40) embryonic stem (ES) cells revealed impaired erythroid differentiation, which was accompanied by a failure to upregulate Scl when erythropoiesis was initiated. Map17 expression was also reduced in hematopoietic tissues and differentiating ES cells, and the Scl +40 element was able to enhance activity of the Map17 promoter. However, only Scl but not Map17 could rescue the Scl(Δ40/Δ40) ES phenotype. Together, these data demonstrate that the Scl +40 enhancer is an erythroid cell-specific enhancer that regulates the expression of both Scl and Map17. Moreover, deletion of the +40 enhancer causes a novel erythroid phenotype, which can be rescued by ectopic expression of Scl but not Map17.

Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages.

The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (Scl(Δ19/Δ19)) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in Scl(Δ19/Δ19) mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in Scl(Δ19/Δ19) fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements.

Mapping and functional characterisation of a CTCF-dependent insulator element at the 3' border of the murine Scl transcriptional domain.

The Scl gene encodes a transcription factor essential for haematopoietic development. Scl transcription is regulated by a panel of cis-elements spread over 55 kb with the most distal 3' element being located downstream of the neighbouring gene Map17, which is co-regulated with Scl in haematopoietic cells. The Scl/Map17 domain is flanked upstream by the ubiquitously expressed Sil gene and downstream by a cluster of Cyp genes active in liver, but the mechanisms responsible for delineating the domain boundaries remain unclear. Here we report identification of a DNaseI hypersensitive site at the 3' end of the Scl/Map17 domain and 45 kb downstream of the Scl transcription start site. This element is located at the boundary of active and inactive chromatin, does not function as a classical tissue-specific enhancer, binds CTCF and is both necessary and sufficient for insulator function in haematopoietic cells in vitro. Moreover, in a transgenic reporter assay, tissue-specific expression of the Scl promoter in brain was increased by incorporation of 350 bp flanking fragments from the +45 element. Our data suggests that the +45 region functions as a boundary element that separates the Scl/Map17 and Cyp transcriptional domains, and raise the possibility that this element may be useful for improving tissue-specific expression of transgenic constructs.

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.

cis-Regulatory remodeling of the SCL locus during vertebrate evolution.

Development progresses through a sequence of cellular identities which are determined by the activities of networks of transcription factor genes. Alterations in cis-regulatory elements of these genes play a major role in evolutionary change, but little is known about the mechanisms responsible for maintaining conserved patterns of gene expression. We have studied the evolution of cis-regulatory mechanisms controlling the SCL gene, which encodes a key transcriptional regulator of blood, vasculature, and brain development and exhibits conserved function and pattern of expression throughout vertebrate evolution. SCL cis-regulatory elements are conserved between frog and chicken but accrued alterations at an accelerated rate between 310 and 200 million years ago, with subsequent fixation of a new cis-regulatory pattern at the beginning of the mammalian radiation. As a consequence, orthologous elements shared by mammals and lower vertebrates exhibit functional differences and binding site turnover between widely separated cis-regulatory modules. However, the net effect of these alterations is constancy of overall regulatory inputs and of expression pattern. Our data demonstrate remarkable cis-regulatory remodelling across the SCL locus and indicate that stable patterns of expression can mask extensive regulatory change. These insights illuminate our understanding of vertebrate evolution.