Vladimir Jankovic

Id1 restrains myeloid commitment, maintaining the self-renewal capacity of hematopoietic stem cells

Appropriate hematopoietic stem cell (HSC) self-renewal reflects the tight regulation of cell cycle entry and lineage commitment. Here, we show that Id1, a dominant-negative regulator of E protein transcription factors, maintains HSC self-renewal by preserving the undifferentiated state. Id1-deficient HSCs show increased cell cycling, by BrdU incorporation in vivo, but fail to efficiently self-renew, leading to low steady-state HSC numbers and premature exhaustion in serial bone marrow transplant assays. The increased cycling reflects the perturbed differentiation process, because Id1 null HSCs more readily commit to myeloid differentiation, with inappropriate expression of myeloerythroid-specific genes. Thus, Id1 appears to regulate the fate of HSCs by acting as a true inhibitor of differentiation.

Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity

RUNX1/AML1 is required for the development of definitive hematopoiesis, and its activity is altered by mutations, deletions, and chromosome translocations in human acute leukemia. RUNX1 function can be regulated by post-translational modifications and protein-protein interactions. We show that RUNX1 is arginine-methylated in vivo by the arginine methyltransferase PRMT1, and that PRMT1 serves as a transcriptional coactivator for RUNX1 function. Using mass spectrometry, and a methyl-arginine-specific antibody, we identified two arginine residues (R206 and R210) within the region of RUNX1 that interact with the corepressor SIN3A and are methylated by PRMT1. PRMT1- dependent methylation of RUNX1 at these arginine residues abrogates its association with SIN3A, whereas shRNA against PRMT1 (or use of a methyltransferase inhibitor) enhances this association. We find arginine-methylated RUNX1 on the promoters of two bona fide RUNX1 target genes, CD41 and PU.1 and show that shRNA against PRMT1 or RUNX1 down-regulates their expression. These arginine methylation sites and the dynamic regulation of corepressor binding are lost in the leukemia-associated RUNX1-ETO fusion protein, which likely contributes to its dominant inhibitory activity.

Id1 Restrains p21 Expression to Control Endothelial Progenitor Cell Formation

Loss of Id1 in the bone marrow (BM) severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis.

Cyclin C Regulates Human Hematopoietic Stem/Progenitor Cell Quiescence†‡§

Hematopoietic stem cells (HSCs) can remain quiescent or they can enter the cell cycle, and either self‐renew or differentiate. Although cyclin C and cyclin dependent kinase (cdk3) are essential for the transition from the G0 to the G1 phase of the cell cycle in human fibroblasts, the role of cyclin C in hematopoietic stem/progenitor cells (HSPCs) is not clear. We have identified an important role of cyclin C (CCNC) in regulating human HSPC quiescence, as knocking down CCNC expression in human cord blood CD34+ cells resulted in a significant increase in quiescent cells that maintain CD34 expression. CCNC knockdown also promotes in vitro HSPC expansion and enhances their engraftment potential in sublethally irradiated immunodeficient mice. Our studies establish cyclin C as a critical regulator of the G0/G1 transition of human HSPCs and suggest that modulating cyclin C levels may be useful for HSC expansion and more efficient engraftment. STEM CELLS 2010;28:308–317

T Cell Recognition of an Engineered MHC Class I Molecule: Implications for Peptide-Independent Alloreactivity

Previously, we described H-2KbW9 (KbW9), an engineered variant of the murine MHC class I molecule H-2Kb (Kb), devoid of the central anchor (“C”) pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of Kb and KbW9 are largely nonoverlapping in vivo. On the basis of these observations, we used KbW9 and Kb to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of KbW9 and Kb. Six of six Kb-specific mAbs, directed against different parts of the molecule, recognized KbW9 well, albeit at different levels than Kb. Furthermore, KbW9 readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, KbW9 mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between Kb and KbW9. Interestingly, when KbW9 was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against Kb and was specific for peptide-independent MHC epitopes. By contrast, Kb-induced alloreactive CTLs recognized Kb in a peptide-specific manner, did not cross-react on KbW9, and were present at much higher frequencies than those induced by KbW9. Thus, induction of rare peptide-independent CTLs depended on unique structural features of KbW9, likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.

Control of hematopoietic stem cell quiescence by the E3 ubiquitin ligase Fbw7

Thompson et al. 2008. J. Exp. Med. doi:10.1084/jem.20080277 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20080277%26rft_id%253Dinfo%253Apmid%252F18474632%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%