Jennifer J. Trowbridge

DNA Methyltransferase 1 Is Essential for and Uniquely Regulates Hematopoietic Stem and Progenitor Cells

DNA methylation is essential for development and in diverse biological processes. The DNA methyltransferase Dnmt1 maintains parental cell methylation patterns on daughter DNA strands in mitotic cells; however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. Defects were observed in Dnmt1-deficient HSC self-renewal, niche retention, and in the ability of Dnmt1-deficient HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had specific impact on myeloid progenitor cells, causing enhanced cell cycling and inappropriate expression of mature lineage genes. Dnmt1 regulates distinct patterns of methylation and expression of discrete gene families in long-term HSCs and multipotent and lineage-restricted progenitors, suggesting that Dnmt1 differentially controls these populations. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment.

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin−c-Kit+Sca-1+ cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.

Hedgehog modulates cell cycle regulators in stem cells to control hematopoietic regeneration.

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here, we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However, this effect is at the expense of HSC function, because continued Hh activation during regeneration represses expression of specific cell cycle regulators, leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo.

Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A.

Reactivation of fetal hemoglobin (HbF) in adults ameliorates the severity of the common β-globin disorders. The transcription factor BCL11A is a critical modulator of hemoglobin switching and HbF silencing, yet the molecular mechanism through which BCL11A coordinates the developmental switch is incompletely understood. Particularly, the identities of BCL11A cooperating protein complexes and their roles in HbF expression and erythroid development remain largely unknown. Here we determine the interacting partner proteins of BCL11A in erythroid cells by a proteomic screen. BCL11A is found within multiprotein complexes consisting of erythroid transcription factors, transcriptional corepressors, and chromatin-modifying enzymes. We show that the lysine-specific demethylase 1 and repressor element-1 silencing transcription factor corepressor 1 (LSD1/CoREST) histone demethylase complex interacts with BCL11A and is required for full developmental silencing of mouse embryonic β-like globin genes and human γ-globin genes in adult erythroid cells in vivo. In addition, LSD1 is essential for normal erythroid development. Furthermore, the DNA methyltransferase 1 (DNMT1) is identified as a BCL11A-associated protein in the proteomic screen. DNMT1 is required to maintain HbF silencing in primary human adult erythroid cells. DNMT1 haploinsufficiency combined with BCL11A deficiency further enhances γ-globin expression in adult animals. Our findings provide important insights into the mechanistic roles of BCL11A in HbF silencing and clues for therapeutic targeting of BCL11A in β-hemoglobinopathies.

Haploinsufficiency of Dnmt1 impairs leukemia stem cell function through derepression of bivalent chromatin domains

Epigenetic mechanisms regulating leukemia stem cells (LSCs) are an attractive target for therapy of blood cancers. Here, we report that conditional knockout of the DNA methyltransferase Dnmt1 blocked development of leukemia, and haploinsufficiency of Dnmt1 was sufficient to delay progression of leukemogenesis and impair LSC self-renewal without altering normal hematopoiesis. Haploinsufficiency of Dnmt1 resulted in tumor suppressor gene derepression associated with reduced DNA methylation and bivalent chromatin marks. These results suggest that LSCs depend on not only active expression of leukemogenic programs, but also DNA methylation-mediated silencing of bivalent domains to enforce transcriptional repression.

Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease

Loss-of-function mutations in the mouse ortholog of C9ORF72 cause fatal autoimmunity that is transferable by bone marrow–derived cells, demonstrating a hematopoietic intrinsic function for the protein encoded by this gene. C9orf72, a suppressor of autoimmunity? Mutations in C9ORF72 are a common contributor to amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of this gene is still poorly defined. In new work, Burberry et al. demonstrate that mutations disrupting the normal function of C9orf72 cause mice to develop features of autoimmunity. They further found that transplantation of normal bone marrow into mutant animals ameliorated this phenotype, whereas transplantation of mutant bone marrow into normal animals was sufficient to cause autoimmunity. The authors conclude that C9orf72 acts through hematopoietic cells to maintain normal immune function and suggest that investigations are warranted into whether disruptions in immunity contribute to disease in patients. C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.

A Single cis Element Maintains Repression of the Key Developmental Regulator Gata2

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element −1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the −1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the −1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed.

Context-dependent function of "GATA switch" sites in vivo.

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the -2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring -1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the -2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

A unique population of bone marrow cells migrates to skeletal muscle via hepatocyte growth factor/c-met axis

Cells expressing the CD45-associated hematopoietic marker are predominantly present in the mammalian bone marrow (BM), but have recently been shown to also reside in the skeletal muscle and potentially participate in muscle repair. Despite the consistent observations, the specific relationship and potential migration of CD45+ cells in the BM versus CD45+ cells residing in the muscle remain unclear, in addition to any understanding of the factors that may regulate the trafficking of CD45+-derived BM cells to skeletal muscle upon i.v. transplantation. Here, transplantation of BM-derived cells fully replaced the CD45+ fraction of skeletal muscle, but gave rise to progenitor cells with distinct hematopoietic lineage capacity from CD45+ cells residing in the BM. Using transwell migration assays, a subset of BM cells was shown to migrate exclusively to mature skeletal muscle cells and not BM-derived stromal cells. Unlike migration of BM cells to stroma, myofiber induced migration of BM-derived cells was not affected by stromal-derived factor-1 (SDF-1) neutralization or CXCR4-blocking antibody, but could be reduced by addition of c-met-blocking antibody and augmented by hepatocyte growth factor (HGF), the putative ligand for c-met. We suggest that the BM compartment consists of a functionally complex population of CD45+ progenitors that includes a subset of HGF/c-met responsive cells capable of migration to skeletal muscle. This previously unappreciated basis for cellular tracking now aids in defining regulatory networks that distinguish the stem cell niche of the BM versus skeletal muscle microenvironments.

DNA methylation in adult stem cells: new insights into self-renewal.

Methylation of cytosine residues in the context of CpG dinucleotides within mammalian DNA is an epigenetic modification with profound effects on transcriptional regulation. A group of enzymes, the DNA methyltransferases (DNMTs) tightly regulate both the initiation and maintenance of these methyl marks. Loss of critical components of this enzymatic machinery results in growth, viability, and differentiation defects in both mice and humans, supporting the notion that this epigenetic modification is essential for proper development. Beyond this, DNA methylation also provides a potent epigenetic mechanism for cellular memory needed to silence repetitive elements and preserve lineage specificity over repeated cell divisions throughout adulthood. Recent work highlighting the specialized roles of DNA methylation and methyltransferases in maintaining adult somatic stem cell function suggests that further dissection of these mechanisms will shed new light on the complex nature of self-renewal.