Brian S. Garrison

High-resolution genome-wide mapping of transposon integration in mammals

ABSTRACT The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon- and virus-based integrating vectors.

Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

The functional potential of hematopoietic stem cells (HSCs) declines during aging, and in doing so, significantly contributes to hematopoietic pathophysiology in the elderly. To explore the relationship between age-associated HSC decline and the epigenome, we examined global DNA methylation of HSCs during ontogeny in combination with functional analysis. Although the DNA methylome is generally stable during aging, site-specific alterations of DNA methylation occur at genomic regions associated with hematopoietic lineage potential and selectively target genes expressed in downstream progenitor and effector cells. We found that age-associated HSC decline, replicative limits, and DNA methylation are largely dependent on the proliferative history of HSCs, yet appear to be telomere-length independent. Physiological aging and experimentally enforced proliferation of HSCs both led to DNA hypermethylation of genes regulated by Polycomb Repressive Complex 2. Our results provide evidence that epigenomic alterations of the DNA methylation landscape contribute to the functional decline of HSCs during aging.

Reprogramming Committed Murine Blood Cells to Induced Hematopoietic Stem Cells with Defined Factors

Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.

Postintegrative Gene Silencing within the Sleeping Beauty Transposition System

ABSTRACT The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates ∼20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative gene silencing, which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2′-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

Summary Hematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

Nf2/Merlin Regulates Hematopoietic Stem Cell Behavior by Altering Microenvironmental Architecture

Stem cell population size is highly regulated across species and tissue types, and alterations are associated with premature tissue failure or cancer. We assessed whether the tumor suppressor and mediator of cell contact inhibition Nf2/merlin plays a role in governing the hematopoietic stem cell pool by stem cell-autonomous or niche-determined processes. Hematopoietic stem cells in Nf2-deficient mice were increased in number and demonstrated a marked shift in location to the circulation. These changes were entirely dependent on changes in the microenvironment, with a marked increase in trabecular bone and marrow vascularity associated with increased VEGF, but without cell-autonomous alterations in stem cell characteristics. Nf2/merlin is critical for maintaining normal structure and function of the hematopoietic stem cell niche. It limits both bone and vascular components, and our model suggests that it thereby constrains stem cell number and position.

Reconstitution in vitro of the V1 complex from the yeast vacuolar proton-translocating ATPase: Assembly recapitulates mechanism

Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V1complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures. Assembly of soluble V1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits. Specifically, V1 can be assembled directly from complex III (subunits E and G) with complex IV (subunits A, B, D, and F) without prior disassembly of complex IV. The formation of complex III in vivo is also shown to be essential and could not be achievedin vitro. Assembly from simpler precursors is possible and is enhanced by added ATP. Assembly can be blocked byN-ethylmaleimide in a Vma1p (subunit A)-specific manner. From these data, we extend our previous model to consider an assembly pathway whose steps reflect the catalytic mechanism of the Boyer binding-change model.

Growth hormone receptor signaling is dispensable for HSC function and aging

Growth hormone receptor (Ghr) signaling is important in a wide variety of cellular processes including aging; however, the role of Ghr signaling in hematopoietic stem cell (HSC) biology remains unexplored. Within the hematopoietic system, Ghr is expressed in a highly HSC-specific manner and is significantly upregulated during aging. Exposure of young and old HSCs to recombinant growth hormone ex vivo led to diminished short-term reconstitution and restored B-cell output from old HSCs. Hematopoietic-specific genetic deletion of Ghr neither impacted steady-state hematopoiesis nor serial transplantation potential. Repeat challenge with 5-fluorouracil showed that Ghr was dispensable for HSC activation and homeostatic recovery in vivo and, after challenge, Ghr-deficient HSCs functioned normally through serial transplantation. Although exogenous Gh induces age-dependent HSC effects, these results indicate that Ghr signaling appears largely dispensable for HSC function and aging.

Reactive Oxygen Species Resulting from Mitochondrial Mutation Diminishes Stem and Progenitor Cell Function

While age-dependent stem cell decline is widely recognized as being a key component of organismal aging, the underlying mechanisms remain elusive. In this issue of Cell Metabolism, Suomalainen and colleagues provide evidence that mitochondrial mutation and associated reactive oxygen species can adversely impact tissue-specific stem and progenitor cell function.