CRISPR-Cas9 Editing of the and Promoters to Treat Sickle Cell Disease.

Background: Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage.

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method.

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of—and molecular basis for—heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes in vivo.

FIAT Deletion Increases Bone Mass But Does Not Prevent High-Fat-Diet-Induced Metabolic Complications.

Factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT) interacts with ATF4 to repress its transcriptional activity. We performed a phenotypic analysis of Fiat-deficient male mice (Fiat-/Y) at 8 and 16 weeks of age. Microcomputed tomography analysis of the distal femur demonstrated 46% and 13% age-dependent increases in trabecular bone volume and thickness, respectively, in Fiat-/Y mice.

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo.

Distinctive Mesenchymal-Parenchymal Cell Pairings Govern B Cell Differentiation in the Bone Marrow.

Bone marrow niches for hematopoietic progenitor cells are not well defined despite their critical role in blood homeostasis. We previously found that cells expressing osteocalcin, a marker of mature osteolineage cells, regulate the production of thymic-seeding T lymphoid progenitors. Here, using a selective cell deletion strategy, we demonstrate that a subset of mesenchymal cells expressing osterix, a marker of bone precursors in the adult, serve to regulate the maturation of early B lymphoid precursors by promoting pro-B to pre-B cell transition through insulin-like growth factor 1 (IGF-1) production.

Heterogeneity of the bone marrow niche.

Purpose Of Review: The bone marrow niche is increasingly recognized as heterogeneous with specific subtypes of mesenchymal niche cells governing the development or homeostasis of selective parenchymal hematopoietic subsets. The present review outlines recent efforts in dissecting these microniches regulated by unique cell pairings within the bone marrow and provides an overview of how the bone marrow orchestrates multiple facets of hematopoiesis.

Recent Findings: Recent advancement in technologies has significantly improved our understanding of the cellular and molecular constituents that contribute to regulation of hematopoiesis and to maintenance of the hematopoietic stem cells (HSCs).

Transcriptome comparison of distinct osteolineage subsets in the hematopoietic stem cell niche using a triple fluorescent transgenic mouse model.

The bone marrow niche is recognized as a central player in maintaining and regulating the behavior of hematopoietic stem and progenitor cells. Specific gain-of and loss-of function experiments perturbing a range of osteolineage cells or their secreted proteins had been shown to affect stem cell maintenance (Calvi et al, 2003 [1]; Stier et al., 2005 [2]; Zhang et al.

Global transcriptome analysis of T-competent progenitors in the bone marrow.

T cells are known to develop in the thymus. However, molecular events that control the transition from hematopoietic progenitor cells in the bone marrow to T precursor cells seeded in the thymus remained poorly defined. Our recent report showed that osteocalcin (Ocn)-expressing bone cells in the bone marrow have major impact on T cell immunity by regulating T progenitor development in the bone marrow (Yu et al.

Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.

Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function.

Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention.

Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches.

Sex steroid blockade enhances thymopoiesis by modulating Notch signaling.

Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner.

Cell-state-specific metabolic dependency in hematopoiesis and leukemogenesis.

The balance between oxidative and nonoxidative glucose metabolism is essential for a number of pathophysiological processes. By deleting enzymes that affect aerobic glycolysis with different potencies, we examine how modulating glucose metabolism specifically affects hematopoietic and leukemic cell populations. We find that a deficiency in the M2 pyruvate kinase isoform (PKM2) reduces the levels of metabolic intermediates important for biosynthesis and impairs progenitor function without perturbing hematopoietic stem cells (HSCs), whereas lactate dehydrogenase A (LDHA) deletion significantly inhibits the function of both HSCs and progenitors during hematopoiesis.

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion.

Altered gene dosage confirms the genetic interaction between FIAT and αNAC.

Factor inhibiting ATF4-mediated transcription (FIAT) interacts with Nascent polypeptide associated complex and coregulator alpha (αNAC). In cultured osteoblastic cells, this interaction contributes to maximal FIAT-mediated inhibition of Osteocalcin (Ocn) gene transcription. We set out to demonstrate the physiological relevance of this interaction by altering gene dosage in compound Fiat and Naca (encoding αNAC) heterozygous mice.

FIAT is co-expressed with its dimerization target ATF4 in early osteoblasts, but not in osteocytes.

FIAT represses osteocalcin gene transcription by heterodimerizing with ATF4 and preventing it from binding to DNA. We report here the expression profiles of FIAT and ATF4 during osteoblastogenesis. Messenger RNA levels for the osteoblast transcriptional regulators Satb2, Runx2, Fiat, and Atf4 were quantified using real-time reverse-transcription PCR (RT-qPCR) and respective protein levels monitored by immunodetection in differentiating primary osteoblast cultures.

FIAT inhibition increases osteoblast activity by modulating Atf4-dependent functions.

The ATF4 transcription factor is a key regulator of osteoblast differentiation that controls osteocalcin gene transcription and type I collagen protein synthesis. We have characterized factor-inhibiting ATF4-mediated transcription (FIAT), a leucine zipper protein that dimerizes with ATF4 to form inactive dimers that cannot bind DNA. Overexpression of FIAT in osteoblasts of transgenic mice inhibited osteocalcin gene transcription and reduced osteoblastic activity, leading to osteopenia (Yu et al.

FIAT represses bone matrix mineralization by interacting with ATF4 through its second leucine zipper.

We have characterized FIAT, a 66 kDa leucine zipper (LZ) protein that dimerizes with activating transcription factor 4 (ATF4) to form inactive dimers that cannot bind DNA. Computer analysis identifies three putative LZ motifs within the FIAT amino acid sequence. We have used deletion- and/or site-specific mutagenesis to individually inactivate these motifs in order to identify the functional LZ that mediates the FIAT-ATF4 interaction.

Identification and transcription of transfer RNA genes in dinoflagellate plastid minicircles.

The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene (psbD, psbE, petD) and one that appears to be an "empty" circle.

Inhibition of ATF4 transcriptional activity by FIAT/gamma-taxilin modulates bone mass accrual.

The basic domain-leucine zipper protein, activating transcription factor 4 (ATF4), was recently shown to control key aspects of osteoblast biology. ATF4 regulates the timely onset of osteoblast differentiation, the synthesis of type I collagen, and the transcription of the osteocalcin and RANKL (receptor activator of NFkappa-B ligand) genes. Accordingly, the levels and activity of ATF4 are under tight control through mechanisms that include protein stability and phosphorylation.

FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic mice.

We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones.