Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis.

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

GATA1 mutations in red cell disorders.

GATA1 is an essential regulator of erythroid cell gene expression and maturation. In its absence, erythroid progenitors are arrested in differentiation and undergo apoptosis. Much has been learned about GATA1 function through animal models, which include genetic knockouts as well as ones with decreased levels of expression. However, even greater insights have come from the finding that a number of rare red cell disorders, including Diamond-Blackfan anemia, are associated with GATA1 mutations. These mutations affect the amino-terminal zinc finger (N-ZF) and the amino-terminus of the protein, and in both cases can alter the DNA-binding activity, which is primarily conferred by the third functional domain, the carboxyl-terminal zinc finger (C-ZF). Here we discuss the role of GATA1 in erythropoiesis with an emphasis on the mutations found in human patients with red cell disorders.

Genome and epigenome analysis of monozygotic twins discordant for congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the leading non-infectious cause of death in infants. Monozygotic (MZ) twins share nearly all of their genetic variants before and after birth. Nevertheless, MZ twins are sometimes discordant for common complex diseases. The goal of this study is to identify genomic and epigenomic differences between a pair of twins discordant for a form of congenital heart disease, double outlet right ventricle (DORV). RESULTS: A monoamniotic monozygotic (MZ) twin pair discordant for DORV were subjected to genome-wide sequencing and methylation analysis. We identified few genomic differences but 1566 differentially methylated regions (DMRs) between the MZ twins. Twenty percent (312/1566) of the DMRs are located within 2 kb upstream of transcription start sites (TSS), containing 121 binding sites of transcription factors. Particularly, ZIC3 and NR2F2 are found to have hypermethylated promoters in both the diseased twin and additional patients suffering from DORV. CONCLUSIONS: The results showed a high correlation between hypermethylated promoters at ZIC3 and NR2F2 and down-regulated gene expression levels of these two genes in patients with DORV compared to normal controls, providing new insight into the potential mechanism of this rare form of CHD.

The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of a defined set of transcription factors requires epigenetic changes in pluripotency genes. Nuclear reprogramming is an inefficient process and the molecular mechanisms that reset the epigenetic state during iPSC generation are largely unknown. Here, we show that downregulation of the nucleosome remodeling and deacetylation (NuRD) complex is required for efficient reprogramming. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs by establishing heterochromatic features and silencing embryonic stem cell-specific marker genes, including Oct4 and Nanog. Depletion of Mbd3, on the other hand, improves reprogramming efficiency and facilitates the formation of pluripotent stem cells that are capable of generating viable chimeric mice, even in the absence of c-Myc or Sox2. The results establish Mbd3/NuRD as an important epigenetic regulator that restricts the expression of key pluripotency genes, suggesting that drug-induced downregulation of Mbd3/NuRD may be a powerful means to improve the efficiency and fidelity of reprogramming.

Building Synthetic Biosensors Using Red Blood Cell Proteins.

As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Toward addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC-protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including noninvasive monitoring of physiological signals for a range of diagnostic applications.

Use of Transesophageal Echocardiography for Liver Transplantation: A Global Comparison of Practice From the ILTS, SATA, and LICAGE.

BACKGROUND: Anesthesiologists frequently use intraoperative transesophageal echocardiography (TEE) to aid in the diagnosis and management of hemodynamic problems during liver transplantation (LT). Although the use of TEE in US centers continues to increase, data regarding international use are lacking. METHODS: This prospective, global, survey-based study evaluates international experience with TEE for LT. Responses from 252 LT (105 US and 147 non-US) centers representing 1789 anesthesiologists were analyzed. RESULTS: Routine use of TEE in the United States has increased in the last 5 y (from 37% to 47%), but only 21% of non-US LT anesthesiologists use TEE routinely. Lack of training (44% US versus 70% non-US) and equipment (9% non-US versus 34% US) were cited as obstacles. Most survey participants preferred not to perform a complete cardiac examination but rather use only 6 of 11 basic views. Although non-US LT anesthesiologists more frequently had additional clinical training than their US counterparts, they had less TEE experience (13% versus 44%) and less frequently, TEE certification (22% versus 35%). Most LT anesthesiologists agreed that TEE certification is essential for proficiency. Of all respondents, 89% agreed or strongly agreed that TEE provides valuable information needed for immediate clinical decision-making, and >86% agreed or strongly agreed that that information could not be derived from other sources. CONCLUSIONS: The use of TEE for LT surgery in the US LT centers is currently higher compared with non-US LT centers. This may become a standard monitoring modality during LT in the near future.

CHD4/NuRD maintains demethylation state of rDNA promoters through inhibiting the expression of the rDNA methyltransferase recruiter TIP5.

Despite the well-established fact that NuRD (nucleosome remodeling and histone deacetylase) is incapable of actively demethylating DNA, the complex is surprisingly showed to be required for the establishment of unmethylated state at promoters of ribosomal genes. But the molecular mechanism underlying how NuRD mediates unmethylation at rDNA promoters remains obscure. Here we show that NuRD directly binds to the promoter of rDNA transcription silencer TIP5 (TTF-I interacting protein 5), one of the components of nucleolar remodeling complex NoRC that silences rRNA genes by recruiting DNA methyltransferase to rDNA promoters and increasing DNA methylation. NuRD negatively regulates TIP5 expression, thereby inhibiting rDNA methylation and maintaining demethylation state of rDNA promoters. The deficiency of NuRD components in reprogrammed cells activates TIP5 expression, resulting in the increased fraction of heterochromatic rRNA genes and transcriptional silencing. Thus, NuRD is able to control methylation status of rDNA promoters through crosstalking with NoRC complex.

Gata1s mutant mice display persistent defects in the erythroid lineage.

GATA1 mutations that result in loss of the N-terminal 83 amino acids are a feature of myeloid leukemia in children with Down syndrome, rare familial cases of dyserythropoietic anemia, and a subset of cases of Diamond-Blackfan anemia. The Gata1s mouse model, which expresses only the short GATA1 isoform that begins at methionine 84, has been shown to have a defect in hematopoiesis, especially impaired erythropoiesis with expanded megakaryopoiesis, during gestation. However, these mice reportedly did not show any postnatal phenotype. Here, we demonstrate that Gata1s mutant mice display macrocytic anemia and features of aberrant megakaryopoiesis throughout life, culminating in profound splenomegaly and bone marrow fibrosis. These data support the use of this animal model for studies of GATA1 deficiencies.

Building synthetic biosensors using red blood cell proteins.

As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Towards addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including non-invasive monitoring of physiological signals for a range of diagnostic applications.

International Liver Transplantation Society/Society for Advancement of Transplant Anesthesia Consensus Statement on Essential Attributes of a Liver Transplant Anesthesiologist.

BACKGROUND: We sought to establish consensus on the essential skills, knowledge, and attributes that a liver transplant (LT) anesthesiologist should possess in a bid to help guide the further training process. METHODS: Consensus was achieved via a modified Delphi methodology, surveying 15 identified international experts in the fields of LT anesthesia and critical care. RESULTS: Key competencies were identified in preoperative management and optimization of a potential LT recipient; intraoperative management, including hemodynamic monitoring; coagulation and potential crisis management; and postoperative intensive and enhanced recovery care. CONCLUSIONS: This article provides an essential guide to competency-based training of an LT anesthesiologist.

LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307.

Use of GoPro point-of-view camera in intubation simulation-A randomized controlled trial.

INTRODUCTION: Teaching endotracheal intubation is uniquely challenging due to its technical, high-stakes, and highly time-sensitive nature. The GoPro is a small, lightweight, high-resolution action camera with a wide-angle field of view that can encompass both the airway as well as the procedurist's hands and positioning technique when worn with a head mount. We aimed to evaluate its effectiveness in improving intubation teaching for novice learners in a simulated setting, via a two-arm, parallel group, randomized controlled superiority trial with 1:1 allocation ratio. METHODS: We recruited Year 4 medical students at the start of their compulsory 2-week Anesthesia posting. Participants underwent a standardized intubation curriculum and a formative assessment, then randomized to receive GoPro or non-GoPro led feedback. After a span of three months, participants were re-assessed in a summative assessment by blinded accessors. Participants were also surveyed on their learning experience for a qualitative thematic perspective. The primary outcomes were successful intubation and successful first-pass intubation. RESULTS: Seventy-one participants were recruited with no dropouts, and all were included in the analysis. 36 participants received GoPro led feedback, and 35 participants received non-GoPro led feedback. All participants successfully intubated the manikin. No statistically significant differences were found between the GoPro group and the non-GoPro group at summative assessment (85.3% vs 90.0%, p = 0.572). Almost all participants surveyed found the GoPro effective for their learning (98.5%). Common themes in the qualitative analysis were: the ability for an improved assessment, greater identification of small details that would otherwise be missed, and usefulness of the unique point-of-view footage in improving understanding. CONCLUSIONS: The GoPro is a promising tool for simulation-based intubation teaching. There are considerations in its implementation to maximize the learning experience and yield from GoPro led feedback and training.

GATA1 insufficiencies in primary myelofibrosis and other hematopoietic disorders: consequences for therapy.

INTRODUCTION: GATA1, the founding member of a family of transcription factors, plays important roles in the development of hematopoietic cells of several lineages. Although loss of GATA1 has been known to impair hematopoiesis in animal models for nearly 25 years, the link between GATA1 defects and human blood diseases has only recently been realized. Areas covered: Here the current understanding of the functions of GATA1 in normal hematopoiesis and how it is altered in disease is reviewed. GATA1 is indispensable mainly for erythroid and megakaryocyte differentiation. In erythroid cells, GATA1 regulates early stages of differentiation, and its deficiency results in apoptosis. In megakaryocytes, GATA1 controls terminal maturation and its deficiency induces proliferation. GATA1 alterations are often found in diseases involving these two lineages, such as congenital erythroid and/or megakaryocyte deficiencies, including Diamond Blackfan Anemia (DBA), and acquired neoplasms, such as acute megakaryocytic leukemia (AMKL) and the myeloproliferative neoplasms (MPNs). Expert commentary: Since the first discovery of GATA1 mutations in AMKL, the number of diseases that are associated with impaired GATA1 function has increased to include DBA and MPNs. With respect to the latter, we are only just now appreciating the link between enhanced JAK/STAT signaling, GATA1 deficiency and disease pathogenesis.

The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.