Kevin M. Pawlik

KLF1 regulates BCL11A expression and [gamma]- to [beta]-globin gene switching

We show that knockdown of KLF1 in human and mouse adult erythroid progenitors markedly reduces BCL11A levels and increases human γ-globin/β-globin expression ratios. These results suggest that KLF1 controls globin gene switching by directly activating β-globin and indirectly repressing γ-globin gene expression. Controlled knockdown of KLF1 in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with β-thalassemia or sickle cell disease.

Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus‐1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self‐inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3′ long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage‐specific embryonic antigen‐1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high‐level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291‐bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic “hit and run” vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells. Stem Cells 2009;27:1042–1049

SNO-hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation

The coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this oxygen-sensing–NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin β-chain (βCys93) and, specifically, for S-nitrosation of βCys93 to form S-nitrosohemoglobin (SNO-Hb). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years. This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the βCys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell–mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of βCys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood cell–dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO bioactivity in vivo.

PRC2 Complexes with JARID2, MTF2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprograming

Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2‐mediated H3K27 methylation and result in the expression of differentiation‐associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4‐mediated reprograming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprograming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage‐associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyltransferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2s activity and gene expression both in ES cells and during somatic cell reprograming. STEM CELLS 2011;29:229–240

Differential Binding of Erythroid Krupple-like Factor to Embryonic/Fetal Globin Gene Promoters during Development

The competition model for β-like globin gene switching during development predicts that differential binding of transcription factors to globin gene promoters and/or proximal enhancers regulate the competitive interactions of globin gene family members with the powerful locus control region (LCR). Direct interactions of individual genes with the LCR are essential for high level expression in erythroid cells. In this paper, we have demonstrated, by chromatin immunoprecipitation, that erythroid-Krupple-like factor (EKLF) binds to embryonic/fetal globin gene promoters in primitive (but not in definitive) erythroid cells. EKLF binds strongly to adult globin gene promoters and to LCR sequences HS4, HS3, HS2, and HS1 in both primitive and definitive erythroid cells. Trimethylation of histone H3K4 and H3K27 at the embryonic/fetal and adult globin gene promoters is equivalent in definitive cells; therefore, the differential binding of EKLF to these promoters does not appear to result from changes in chromatin configuration. Interestingly, the level of EKLF in definitive cells is 3-fold higher than the level in primitive cells. These results suggest that temporal-specific changes in EKLF abundance result in differential binding of this essential erythroid transcription factor to embryonic/fetal globin gene promoters during development and that these changes in EKLF binding specificity mediate the competitive interactions of globin gene family members with the LCR.

Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting

Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.

SRY (sex determining region Y)-box2 (Sox2)/poly ADP-ribose polymerase 1 (Parp1) complexes regulate pluripotency

To gain insight into mechanisms controlling SRY (sex determining region Y)-box 2 (Sox2) protein activity in mouse embryonic stem cells (ESCs), the endogenous Sox2 gene was tagged with FLAG/Hemagglutinin (HA) sequences by homologous recombination. Sox2 protein complexes were purified from Sox2/FLAG/HA knockin ESCs, and interacting proteins were defined by mass spectrometry. One protein in the complex was poly ADP-ribose polymerase I (Parp1). The results presented below demonstrate that Parp1 regulates Sox2 protein activity. In response to fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling, Parp1 auto-poly ADP-ribosylation enhances Sox2-Parp1 interactions, and this complex inhibits Sox2 binding to octamer-binding transcription factor 4 (Oct4)/Sox2 enhancers. Based on these results, we propose a unique mechanism in which FGF signaling fine-tunes Sox2 activity through posttranslational modification of a critical interacting protein, Parp1, and balances the maintenance of ESC pluripotency and differentiation. In addition, we demonstrate that regulation of Sox2 activity by Parp1 is critical for efficient generation of induced pluripotent stem cells.

The AS‐RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation

Antisense RNAs regulate the transcription and translation of the corresponding sense genes. Here, we report that an antisense RNA, AS‐RBM15, is transcribed in the opposite direction within exon 1 of RBM15. RBM15 is a regulator of megakaryocyte (MK) differentiation and is also involved in a chromosome translocation t(1;22) in acute megakaryocytic leukemia. MK terminal differentiation is enhanced by up‐regulation of AS‐RBM15 expression and attenuated by AS‐RBM15 knockdown. At the molecular level, AS‐RBM15 enhances RBM15 protein translation in a CAP‐dependent manner. The region of the antisense AS‐RBM15 RNA, which overlaps with the 5′UTR of RBM15, is sufficient for the up‐regulation of RBM15 protein translation. In addition, we find that transcription of both RBM15 and AS‐RBM15 is activated by the transcription factor RUNX1 and repressed by RUNX1‐ETO, a leukemic fusion protein. Therefore, AS‐RBM15 is a regulator of megakaryocyte differentiation and may play a regulatory role in leukemogenesis.

Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (βA/[βS+βA]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications.

Serine Threonine Kinase Receptor‐Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1‐Mediated Retinoic Acid Homeostasis

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD‐domain protein STRAP (serine threonine kinase receptor‐associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel‐like factor 9 (KLF9) binding motifs to be critical in the enhancement of non‐canonical RA‐induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior‐posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368–1379