No abstract

SUMMARY GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction, and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for cross-regulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2 negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy.C ellular differentiation requires massive remodeling of subcellular structures to accommodate specialized functions of cell progeny. For example, the differentiation of committed hematopoietic progenitors into erythrocytes requires disposal of mitochondria (mitophagy) and nuclei (enucleation), which are not required for erythrocyte function. The process whereby cells consume organelles is termed autophagy (29). Autophagy mediates morphological remodeling in developmental and pathophysiological contexts. A cohort of autophagy proteins functions in a multistep reaction to generate an autophagosome that engulfs damaged organelles (53). The subsequent fusion of the loaded autophagosome with the lysosome results in proteolysis of the engulfed proteins.Core components of the autophagy machinery are broadly expressed, and therefore diverse cell types are competent to execute autophagy. A recent proteomics analysis expanded the repertoire of proteins linked to autophagy and illustrated their complex interaction networks (2). Many questions remain unanswered regarding how this complex network of apparently ubiquitous autophagy components is established and what role cell-type-specific factors have in instigating and regulating autophagy.Since autophagy is a critical step in erythrocyte development (19,39,41,57), it is particularly instructive to analyze cell-typespecific autophagy in this context. Targeted deletion of the BCL-2 family member NIX yields anemia, impaired erythroid maturation, and impaired mitophagy during terminal erythroid differentiation (39, 41). Furthermore, a conditional knockout in hematopoietic cells of Atg7, which encodes a protein resembling a ubiquitin-activating enzyme, yields severe anemia, defective erythropoiesis, and lethality shortly af...

Within the sperm nucleus the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such they may soon be utilized as clinical markers of male fertility. In this review we explore and discuss how this may be orchestrated.

Vascular endothelial dysfunction underlies the genesis and progression of numerous diseases. Although the GATA transcription factor GATA-2 is expressed in endothelial cells and is implicated in coronary heart disease, it has been studied predominantly as a master regulator of hematopoiesis. Because many questions regarding GATA-2 function in the vascular biology realm remain unanswered, we used ChIP sequencing and loss-of-function strategies to define the GATA-2–instigated genetic network in human endothelial cells. In contrast to erythroid cells, GATA-2 occupied a unique target gene ensemble consisting of genes encoding key determinants of endothelial cell identity and inflammation. GATA-2–occupied sites characteristically contained motifs that bind activator protein-1 (AP-1), a pivotal regulator of inflammatory genes. GATA-2 frequently occupied the same chromatin sites as c-JUN and c-FOS, heterodimeric components of AP-1. Although all three components were required for maximal AP-1 target gene expression, GATA-2 was not required for AP-1 chromatin occupancy. GATA-2 conferred maximal phosphorylation of chromatin-bound c-JUN at Ser-73, which stimulates AP-1–dependent transactivation, in a chromosomal context-dependent manner. This work establishes a link between a GATA factor and inflammatory genes, mechanistic insights underlying GATA-2–AP-1 cooperativity and a rigorous genetic framework for understanding GATA-2 function in normal and pathophysiological vascular states.

Because obesity rates have increased dramatically over the past 3 decades, type 2 diabetes has become increasingly prevalent as well. Type 2 diabetes is associated with decreased pancreatic β-cell mass and function, resulting in inadequate insulin production. Conversely, in nondiabetic obesity, an expansion in β-cell mass occurs to provide sufficient insulin and to prevent hyperglycemia. This expansion is at least in part due to β-cell proliferation. This review focuses on the mechanisms regulating obesity-induced β-cell proliferation in humans and mice. Many factors have potential roles in the regulation of obesity-driven β-cell proliferation, including nutrients, insulin, incretins, hepatocyte growth factor, and recently identified liver-derived secreted factors. Much is still unknown about the regulation of β-cell replication, especially in humans. The extracellular signals that activate proliferative pathways in obesity, the relative importance of each of these pathways, and the extent of cross-talk between these pathways are important areas of future study.

It is well established that nuclear architecture plays a key role in poising regions of the genome for transcription. This may be achieved using scaffold/matrix attachment regions (S/MARs) that establish loop domains. However, the relationship between changes in the physical structure of the genome as mediated by attachment to the nuclear scaffold/matrix and gene expression is not clearly understood. To define the role of S/MARs in organizing our genome and to resolve the often contradictory loci-specific studies, we have surveyed the S/MARs in HeLa S3 cells on human chromosomes 14–18 by array comparative genomic hybridization. Comparison of LIS (lithium 3,5-diiodosalicylate) extraction to identify SARs and 2 m NaCl extraction to identify MARs revealed that approximately one-half of the sites were in common. The results presented in this study suggest that SARs 5′ of a gene are associated with transcript presence whereas MARs contained within a gene are associated with silenced genes. The varied functions of the S/MARs as revealed by the different extraction methods highlights their unique functional contribution.