C. Scott Swindle

Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal–regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-“tethered” ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of “insoluble” growth factor ligands and a novel mode of activation for growth factor receptors.

Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells.

Although DNA methylation and transcriptional repression are generally associated, a causal role for DNA methylation in silencing of retroviral vectors has not been established. The newer generation murine stem cell virus retroviral vector (MSCV) lacks many of the repressive cis-acting DNA sequences identified in Moloney murine leukemia virus but remains sensitive to transcriptional silencing in various cell types. To determine the contribution of cytosine methylation to MSCV silencing, we mutated CpG dinucleotides located in the MSCV long terminal repeat (LTR) that are clustered in the U3 region and directly spanning the transcription start site in the R region. Effects of the CpG mutations on MSCV silencing were assessed in murine embryonic stem cells. An analysis of numerous clonal proviral integrants showed that mutation of CpGs in both clusters eliminated proviral integrants that were completely silenced. Variegated expression was shown to represent a substantial component of intraclonal silencing and was independent of the presence of CpGs in the LTR. Treatment of transduced cells with 5-azadeoxycytidine delayed establishment of the silenced state but had only a modest effect on expression of some proviral integrants at late times post-transduction. These results are direct evidence for a causal contribution of DNA methylation in the LTR to MSCV silencing and define the promoter region CpGs as a repressive element in embryonic stem cells. Furthermore, distinct mechanisms are suggested for establishment and maintenance of the silenced proviral state.

Activated Notch2 Potentiates CD8 Lineage Maturation and Promotes the Selective Development of B1 B Cells

ABSTRACT Although studies have shown that the Notch2 family member is critical for embryonic development, little is known concerning its role in hematopoiesis. In this study, we show that the effects of an activated form of Notch2 (N2IC) on the T-cell lineage are dosage related. High-level expression of N2IC results in the development of T-cell leukemias. In contrast, lower-level expression of N2IC does not lead to transformation but skews thymocyte development to the CD8 lineage. Underlying this skew is a dramatic enhancement in positive selection and CD8SP maturation. N2IC permits early B-cell development but blocks the maturation of conventional B2 cells at the pre-B stage, which is the limit of endogenous Notch2 protein expression in developing B cells. Most strikingly, while B2 B cell development is blocked at the pre-B-cell stage, N2IC promotes the selective development of LPS-responsive B1 B cells. This study implicates a role for Notch2 in the maturation of the CD8 lineage and suggests a novel function for Notch2 in the development of the B1 B-cell subset.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

Significance Polycomb repressive complex 1 (PRC1) represents an important epigenetic regulator, which exerts its effect on gene expression via histone H2A ubiquitination (ubH2A). We developed a conditional Usp16 knockout mouse model and demonstrated that Usp16 is indispensable for hematopoiesis and hematopoietic stem cell (HSC) lineage commitment. We identified Usp16 to be a H2A deubiquitinase that counterbalances the PRC1 ubiquitin ligase to control ubH2A level in the hematopoietic system. Conditional Usp16 deletion led to altered expression of many regulators of chromatin organization and hematopoiesis. In addition, Usp16 maintains normal HSC cell cycle status via repressing the expression of Cdkn1a, which encodes p21cip1, an inhibitor of cell cycle entry. This study provides novel insights into the epigenetic mechanism that regulates hematopoiesis and HSC function. Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Enhanced hematopoietic stem cell self-renewal-promoting ability of clonal primary MSC versus their osteogenic progeny

Long‐term self‐renewing hematopoietic stem cell (LT‐HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT‐HSC and a number of niche‐associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin+Lepr +Sca‐1+CD146+ and could be stably propagated with high colony‐forming unit fibroblast re‐cloning efficiency. MSC synergized with SCF and IL‐11 to support a 20‐fold expansion in true LT‐HSC after 10‐days of in vitro coculture. Optimal stimulation of LT‐HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self‐renewal‐promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT‐HSC homeostasis in vivo. Stem Cells 2017;35:473–484

Allogeneic Th1 Cells Home to Host Bone Marrow and Spleen and Mediate IFNγ-Dependent Aplasia

Bone marrow graft failure and poor graft function are frequent complications after hematopoietic stem cell transplantation and result in significant morbidity and mortality. Both conditions are associated with graft-versus-host disease (GVHD), although the mechanism remains undefined. Here we show, in 2 distinct murine models of GVHD (complete MHC- and class II-disparate) that mimic human peripheral blood stem cell transplantation, that Th1 CD4(+) cells induce bone marrow failure in allogeneic recipients. Bone marrow failure after transplantation of allogeneic naïve CD4(+) T cells was associated with increased CD4(+) Th1 cell development within bone marrow and lymphoid tissues. Using IFNγ-reporter mice, we found that Th1 cells generated during GVHD induced bone marrow failure after transfers into secondary recipients. Homing studies demonstrated that transferred Th1 cells express CXCR4, which was associated with accumulation within bone marrow and spleen. Allogeneic Th1 cells were activated by radiation-resistant host bone marrow cells and induced bone marrow failure through an IFNγ-dependent mechanism. Thus, allogeneic Th1 CD4(+) cells generated during GVHD traffic to hematopoietic sites and induce bone marrow failure via IFNγ-mediated toxicity. These results have important implications for prevention and treatment of bone marrow graft failure after hematopoietic stem cell transplantation.