Yvette Y. Yien

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

Background: CRISPR/Cas9-directed cleavages may result in genomic deletion. Results: CRISPR/Cas9-produced genomic deletion frequency is inversely related to deletion size, with large deletions and inversions practicable and biallelic deletions exceeding probabilistic expectation. Conclusion: Biallelic, large genomic deletions are efficiently engineered in mammalian cells by CRISPR/Cas9. Significance: CRISPR/Cas9-mediated genomic deletion represents a robust method for loss-of-function studies in mammalian cells. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.

Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis

The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is 5-fold reduced in yeast lacking mtClpX activity and that total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX, therefore, is a widely conserved stimulator of an essential biosynthetic pathway and uses a previously unrecognized mechanism for AAA+ unfoldases.

TMEM14C is required for erythroid mitochondrial heme metabolism

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals

Shipping iron around in small packages Iron plays a crucial role in a wide variety of biological functions, which in turn rely on the proteins that transport the metal in and out of cells. Grillo et al. used a simple lipophilic small molecule that binds iron ions to restore transport in animal models with deficiencies in iron transporters. This cyclic ketol, hinokitiol, was first tested in yeast and then shown to promote gut iron absorption in rats and mice, as well as hemoglobin production in zebrafish. Science, this issue p. 608 A simple lipophilic compound restores iron transport in and out of cells in the absence of proteins with that function. Multiple human diseases ensue from a hereditary or acquired deficiency of iron-transporting protein function that diminishes transmembrane iron flux in distinct sites and directions. Because other iron-transport proteins remain active, labile iron gradients build up across the corresponding protein-deficient membranes. Here we report that a small-molecule natural product, hinokitiol, can harness such gradients to restore iron transport into, within, and/or out of cells. The same compound promotes gut iron absorption in DMT1-deficient rats and ferroportin-deficient mice, as well as hemoglobinization in DMT1- and mitoferrin-deficient zebrafish. These findings illuminate a general mechanistic framework for small molecule–mediated site- and direction-selective restoration of iron transport. They also suggest that small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases.

Functional Interactions between Erythroid Kruppel-like Factor (EKLF/KLF1) and Protein Phosphatase PPM1B/PP2Cβ

Background: EKLF is a transcription factor that is critical for erythroid gene expression. Results: The EKLF in vivo interaction with Ppm1b phosphatase has been identified after co-immunoprecipitation. Conclusion: The effect of the interaction is to modulate EKLF stability and activity. Significance: Our data uncover a novel interaction and reveal a multilayered and contextual difference in how Ppm1b alters EKLF regulation of its downstream targets. Erythroid Krüppel-like factor (EKLF; KLF1) is an erythroid-specific transcription factor required for the transcription of genes that regulate erythropoiesis. In this paper, we describe the identification of a novel EKLF interactor, Ppm1b, a serine-threonine protein phosphatase that has been implicated in the attenuation of NFκB signaling and the regulation of Cdk9 phosphorylation status. We show that Ppm1b interacts with EKLF via its PEST1 sequence. However, its genetic regulatory role is complex. Using a promoter-reporter assay in an erythroid cell line, we show that Ppm1b superactivates EKLF at the β-globin and BKLF promoters, dependent on intact Ppm1b phosphatase activity. Conversely, depletion of Ppm1b in CD34+ cells leads to a higher level of endogenous β-globin gene activation after differentiation. We also observe that Ppm1b likely has an indirect role in regulating EKLF turnover via its zinc finger domain. Together, these studies show that Ppm1b plays a multilayered role in regulating the availability and optimal activity of the EKLF protein in erythroid cells.

Erratum: Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells (The Journal of Biological Chemistry (2014) 289 (21312-21324) DOI: 10.1074/jbc.M114.564625)

Matthew C. Canver, Daniel E. Bauer, Abhishek Dass, Yvette Y. Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Barry H. Paw, and Stuart H. Orkin There was an error in the title and the first sentence of the abstract. The CRISPR acronym should be clustered regularly interspaced short palindromic repeats (CRISPR). THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 6, p. 2556, February 10, 2017

Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria

Significance Although heme synthesis is ubiquitous, specific regulatory mechanisms couple heme production to cellular demand and environmental conditions. The importance of these regulatory mechanisms is highlighted by clinical variability in porphyrias caused by loss-of-function mutations in heme synthesis enzymes. Heme synthesis is also controlled by the mitochondrial AAA+ unfoldase ClpX, which participates in both heme-dependent degradation of δ-aminolevulinate synthase (ALAS) and ALAS activation. This study reports a human familial mutation in CLPX that contributes to erythropoietic protoporphyria (EPP) by partially inactivating CLPX. Reduced CLPX activity increases ALAS post-translational stability, causing pathological accumulation of protoporphyrin IX (PPIX) in human patients. Our results thus identify an additional gene that promotes PPIX overproduction and EPP and highlight the complex gene network contributing to disorders of heme metabolism. Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Reductions in the mitochondrial ABC transporter Abcb10 affect the transcriptional profile of heme biosynthesis genes

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10s ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

A role for iron deficiency in dopaminergic neurodegeneration

Disorders of iron metabolism, manifesting in iron overload or iron deficiency, are implicated in neurodegeneration (1⇓⇓⇓–5). In PNAS, Matak et al. (6) report the specific inactivation of ferroportin ( Fpn , Slc40a1 ) and transferrin receptor 1 ( TfR1 ) in dopaminergic (DA) neurons. The animal models generated in this study demonstrated that Fpn did not play a major role in DA neurophysiology, whereas a defect in Tfr1 -dependent iron uptake caused severe iron deficiency that resulted in neurodegeneration, manifesting in behaviors that are similar to those in Parkinson’s disease (6). Iron transporters play a key role in iron homeostasis and tightly couple intracellular iron levels with cellular requirements (7). This Matak et al. (6) study examines the requirement for FPN, the only known cellular iron exporter (8⇓–10), and TFR1, a key component of the iron uptake machinery, to dissect the effects of iron overload and iron deficiency in the biology of DA neurons. Loss of Fpn function in several cell types causes iron overload (11, 12) and has been implicated in myelination defects (13). TfR1 -mediated iron uptake is the main source of iron for actively proliferating cells, and is essential for iron transport in erythroid cells and neural tissue (14), epithelial enterocytes (15), skeletal muscle (16), and cardiac muscle (17), in addition to DA neurons, as described in Matak et al. (6). TfR1 deficiency in erythroid … [↵][1]1To whom correspondence should be addressed. Email: bpaw{at}rics.bwh.harvard.edu. [1]: #xref-corresp-1-1