Ronald G. Nachtman

E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors.

OBJECTIVE FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. MATERIALS AND METHODS FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation. RESULTS Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent. CONCLUSIONS The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.

Murine hematopoietic stem cells and multipotent progenitors express truncated intracellular form of c-kit receptor.

The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors.

Screening, cloning and characterization of novel transcription factors involved in blood cell development

Abstract To identify new hematopoietic transcription factors we have performed a comprehensive degenerate PCR cloning and developmental and tissue-specific expression analysis of 71 Cys 2 -His 2 type zinc finger (ZF) genes expressed in mouse Lin − Sca-1 + bone marrow cells (highly enriched for HSC and primitive progenitors). Based on sequence data and association of expression pattern with specific stages of blood cell development, 4 Lin − Sca-1 + cell-derived ZF genes were selected for functional analysis. Here we describe Vulcan and Tycho , two new ZF genes with tissue-restricted and developmentally regulated expression during hematopoiesis. Vulcan encodes a 355 a protein with a novel type of KRAB A repressor domain and 3 Cys 2 -His 2 ZF motifs at the C-terminus. Vulcan is expressed throughout mouse embryonic development (including fetal liver), and in adult thymus, spleen, thyroid, submaxillary gland, brain and testis. During hematopoiesis, Vulcan is expressed in HSC, pluripotent progenitors, pro-B, pre-B and T cells, but not in HPP-CFC cells and mature myeloid cells. Tycho gene encodes a 425 aa protein with 11 Cys 2 -His 2 ZF domains, which is expressed throughout mouse embryonic development, and at a very low level in adult mouse brain, liver, heart, and testis. Compared to Lin − Sca-1 + cells, transcription of Tycho is up-regulated 2-4-fold in Lin − Sca-1 + cells and pluripotent progenitor cell lines, and 6 to 8-fold in spleen, thymus and T cell lines. HPP-CFC cells, myeloid progenitors, monocytes, macrophages and B lineage cells (pro-B, Pre-B, B) do not express or express very low levels of Tycho . To study the in vivo rule of Vulcan and Tycho in hematopoiesis, we are generating knockout and transgenic mice.

Microinjection of purified transgenic hsc and progenitors into blastocysts leads to partial complementation of t cell compartment in chimeric adult RAG-2 mice

Abstract To further analyze in vivo developmental potential of mouse HSC and progenitors we have developed a new pre-utero transplantation assay termed blastocyst engraftment assay (BEA). Microinjection of ≤ 100 Lin − Sca-1 + or Lin − Sca-1 − cells (purified by FACS from the bone marrow of adult ROSA β- geo 26 transgenic mice) into 3.5 dpc C57BL/6J blastocysts has demonstrated that only the population of more primitive Lin − Sca-1 + cells, but not Lin − Sca-1 − cells, contains blastocyst colonizing cells (BCC), which can survive during embryo development and engraft yolk sac and fetal liver (≤ 5% of transgenic cells). To investigate whether microinjected HSC and progenitors could repopulate lymphoid compartments, we have generated chimeric RAG-2 mice and analyzed complementation level of lymphoid cells in 4 weeks old control and experimental RAG-2 mice by flow cytometry and PCR. Comparative analysis of thymocytes from control and experimental RAG-2 mice has revealed an increase in the number of SP T cells (CD4+CD8− and CD4−CD8+), and appearance of double positive (CD4+CD8+) and TCRαβ 1 cells in the thymus of chimeric mice. Interestingly, analysis of T and B cells in the spleen and bone marrow has not shown complementation in experimental mice. Low engraftment level of donor HSC and partial complementation of T cells compartment are probably due to the lack of selective functional advantage over endogenous RAG-2 −/− HSC. Microinjection of highly enriched populations of HSC into blastocysts of knockout mice (GATA-2, SCL, AML-1, PU-1) with perturbed hematopoiesis could serve as a novel in vivo assay to analyze primitive hematopoietic cell homing and differentiation during early embryonic development.

Cloning and functional characterization of novel genes preferentially expressed in hematopoietic cells

Abstract Elucidation of the temporal and spatial pattern of differential gene expression during differentiation of hematopoietic stem cells and progenitors could have far reaching implications for ex vivo manipulation of HSC, clinical BM transplantation and gene therapy of hematologic disorders. It is likely that combined use of differential screening methods and cDNA microarrays will be necessary to generate a nonredundant database of differentially expressed genes and transcript catalogues for murine and human HSC and progenitors. Our strategy for isolation of genes preferentially expressed in mouse HSC and progenitors was based on (a) differential screening and subtraction of high quality full length cDNA libraries prepared from purified mouse Lin−Sca-1+ and Lin−Sca-1− BM cells, (b) cDNA sequencing, and (c) developmental and tissue-specific expression analysis of selected cDNAs. Here we report cloning of 3 novel genes with preferential expression in hematopoietic cells. Clone LS215 is a novel gene with multiple transmembrane domains, and is expressed in mouse ES cells, embryo, HSC, thymus, lymph node and uterus. Clone LS372 is a novel ring finger gene that is expressed in mouse ES cells, embryo, HSC, thymus and kidney. Clone LS416 is a novel KRAB zinc finger gene that is expressed in mouse ES cells, embryo, HSC, thymus, lymph node and skin. Using ES cell clones in which alleles for LS215, LS372, and LS416 were inactivated by ATG-less gene trap vector pGT1.8 βgeo (Dr. William Skarnes, UC Berkeley), we are generating knockout mice to study the role of these genes during blood cell development and differentiation.