GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors.

Anterior-posterior (A-P) specification of the neural tube involves initial acquisition of anterior fate followed by the induction of posterior characteristics in the primitive anterior neuroectoderm. Several morphogens have been implicated in the regulation of A-P neural patterning; however, our understanding of the upstream regulators of these morphogens remains incomplete. Here, we show that the Krüppel-like zinc finger transcription factor GLI-Similar 3 (GLIS3) can direct differentiation of human embryonic stem cells (hESCs) into posterior neural progenitor cells in lieu of the default anterior pathway. Transcriptomic analyses reveal that this switch in cell fate is due to rapid activation of Wingless/Integrated (WNT) signaling pathway. Mechanistically, through genome-wide RNA-Seq, ChIP-Seq, and functional analyses, we show that GLIS3 binds to and directly regulates the transcription of several WNT genes, including the strong posteriorizing factor WNT3A, and that inhibition of WNT signaling is sufficient to abrogate GLIS3-induced posterior specification. Our findings suggest a potential role for GLIS3 in the regulation of A-P specification through direct transcriptional activation of WNT genes. Stem Cells 2018 Stem Cells 2019;37:202-215.

Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing.

Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.

Intragenic Enhancers Attenuate Host Gene Expression.

Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.

Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors.

Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y's distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways.

A CALM-derived nuclear export signal is essential for CALM-AF10-mediated leukemogenesis.

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

The PICALM protein plays a key role in iron homeostasis and cell proliferation.

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.