Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

Alterations in chromatin play an important role in oncogenic transformation, although the underlying mechanisms are often poorly understood. The SWI/SNF complex contributes to epigenetic regulation by using the energy of ATP hydrolysis to remodel chromatin and thus regulate transcription of target genes. SNF5, a core subunit of the SWI/SNF complex, is a potent tumor suppressor that is specifically inactivated in several types of human cancer. However, the mechanism by which SNF5 mutation leads to cancer and the role of SNF5 within the SWI/SNF complex remain largely unknown. It has been hypothesized that oncogenesis in the absence of SNF5 occurs due to a loss of function of the SWI/SNF complex. Here, we show, however, distinct effects for inactivation of Snf5 and the ATPase subunit Brg1 in primary cells. Further, using both human cell lines and mouse models, we show that cancer formation in the absence of SNF5 does not result from SWI/ SNF inactivation but rather that oncogenesis is dependent on continued presence of BRG1. Collectively, our results show that cancer formation in the absence of SNF5 is dependent on the activity of the residual BRG1-containing SWI/SNF complex. These findings suggest that, much like the concept of oncogene addiction, targeted inhibition of SWI/SNF ATPase activity may be an effective therapeutic approach for aggressive SNF5-deficient human tumors.

Cells remove unstable polypeptides through protein quality-control (PQC) pathways such as ubiquitin-mediated proteolysis and autophagy. In the present study, we investigated how these pathways are used in ␤-thalassemia, a common hemoglobinopathy in which ␤-globin gene mutations cause the accumulation and precipitation of cytotoxic ␣-globin subunits. In ␤-thalassemic erythrocyte precursors, free ␣-globin was polyubiquitinated and degraded by the proteasome. These cells exhibited enhanced proteasome activity, and transcriptional profiling revealed coordinated induction of most proteasome subunits that was mediated by the stress-response transcription factor Nrf1. In isolated thalassemic cells, short-term proteasome inhibition blocked the degradation of free ␣-globin. In contrast, prolonged in vivo treatment of ␤-thalassemic mice with the proteasome inhibitor bortezomib did not enhance the accumulation of free ␣-globin. Rather, systemic proteasome inhibition activated compensatory proteotoxic stress-response mechanisms, including autophagy, which cooperated with ubiquitin-mediated proteolysis to degrade free ␣-globin in erythroid cells. Our findings show that multiple interregulated PQC responses degrade excess ␣-globin. Therefore, ␤-thalassemia fits into the broader framework of protein-aggregation disorders that use PQC pathways as cellprotective mechanisms. (Blood. 2012; 119(22):5265-5275) IntroductionThe production of functional hemoglobin A (HbA) tetramers (␣ 2 ␤ 2 ) requires the coordinated synthesis and assembly of ␣-and ␤-globin protein chains and iron-containing heme groups. Individually, all HbA components are toxic to RBCs and their precursors, as illustrated by ␤-thalassemias, a common hemoglobinopathy in which ␤-globin gene (HBB) mutations cause the buildup of free ␣-globin. 1 These unpaired ␣ chains initiate an oxidative damage cascade and form damaging precipitates that contribute largely to the clinical problems associated with ␤-thalassemia.The pathophysiology of ␤-thalassemia bears similarities to a diverse group of protein-aggregation diseases affecting multiple organs (for review, see Khandros and Weiss 2 ). These disorders, which include Parkinson disease, Alzheimer disease, Huntington disease, amyotrophic lateral sclerosis, and ␣ 1 -antitrypsin deficiency, are caused by the accumulation of unstable, relatively insoluble proteins. It is believed that the affected cells can detoxify and remove these damaging proteins via multiple interacting biochemical pathways called protein quality-control (PQC) pathways, but that disease ensues when such compensatory mechanisms are overwhelmed (for review, see Ciechanover and Brundin,3 Ding and Yin, 4 and Jaeger and Wyss-Coray 5 ). Cellular PQC systems include molecular chaperones, ubiquitin-mediated proteolysis, and autophagy. Several lines of evidence suggest that ␤-thalassemic erythroid cells use PQC pathways to detoxify free ␣-globin (for review, see Khandros and Weiss 2 ): (1) the clinical severity of ␤-thalassemia is proportional to the degree of ␣-glo...

There is a growing appreciation of the role that epigenetic alterations can play in oncogenesis. However, given the large number of genetic anomalies present in most cancers, it has been difficult to evaluate the extent to which epigenetic changes contribute to cancer. SNF5 (INI1/SMARCB1/BAF47) is a tumor suppressor that regulates the epigenome as a core member of the SWI/SNF chromatin remodeling complex. While the SWI/SNF complex displays potent tumor suppressor activity, it is unknown whether this activity is exerted genetically via maintenance of genome integrity or epigenetically via transcriptional regulation. Here we show that Snf5-deficient primary cells do not show altered sensitivity to DNA damaging agents, defects in ␥-H2AX induction, or an abrogated DNA damage checkpoint. Further, the aggressive malignancies that arise following SNF5 loss are diploid and genomically stable. Remarkably, we demonstrate that most human SNF5-deficient cancers lack genomic amplifications/deletions and, aside from SNF5 loss, are indistinguishable from normal cells on single-nucleotide polymorphism arrays. Finally, we show that epigenetically based changes in transcription that occur following SNF5 loss correlate with the tumor phenotype. Collectively, our results provide novel insight into the mechanisms of oncogenesis by demonstrating that disruption of a chromatin remodeling complex can largely, if not completely, substitute for genomic instability in the genesis of aggressive cancer.Many safeguards are in place to prevent a cell from undergoing oncogenic transformation. For example, an average of 14 separate cancer-promoting mutations are required for formation of breast or colon cancer (70). The mechanisms that underlie cancer formation are complex, however, since the low rate of mutation in normal human cells should essentially preclude the independent acquisition of so many mutations. Genome instability may explain this apparent paradox, since early mutations that compromise DNA repair can accelerate the acquisition of subsequent mutations (3). Consistent with this, both DNA repair defects and chromosomal instability are extremely common among highly malignant cancers. However, the cause-and-effect relationships between oncogenesis and genomic instability are not clear, and genetic mutation is not the only mechanism by which stable alteration of gene expression can occur.It has become increasingly clear that epigenetic alterations, those changes in gene expression brought about by altered chromatin context rather than DNA sequence mutations, can also contribute to oncogenic transformation. For example, hypermethylation of DNA in CpG-rich promoter regions can silence tumor suppressor genes. Similarly, expression levels of histone methyltransferases, histone acetyltransferases, and histone deacetylases are frequently altered in cancer cells, as are global levels of histone acetylation and methylation (20,30,67). Recently nucleosome positioning was also implicated in oncogenesis, with the demonstration that heritable cha...

SUMMARY TRIM58 is an E3 ubiquitin ligase superfamily member implicated by genome wide association studies (GWAS) to regulate human erythrocyte traits. Here we show that Trim58 expression is induced during late erythropoiesis and that its depletion by shRNAs inhibits the maturation of late stage nucleated erythroblasts to anucleate reticulocytes. Imaging flow cytometry studies demonstrate that Trim58 regulates polarization and/or extrusion of erythroblast nuclei. In vitro, Trim58 directly binds and ubiquitinates the intermediate chain of the microtubule motor dynein. In cells, Trim58 stimulates proteasome-dependent degradation of the dynein holoprotein complex. During erythropoiesis, Trim58 expression, dynein loss and enucleation occur concomitantly and all are inhibited by Trim58 shRNAs. Dynein regulates nuclear positioning and microtubule organization, both of which undergo dramatic changes during erythroblast enucleation. Thus, we propose that Trim58 regulates this process by eliminating dynein. Our findings identify an erythroid-specific regulator of enucleation and elucidate a previously unrecognized mechanism for controlling dynein activity.

A fundamental goal in cancer research is the identification of the cell types and signaling pathways capable of initiating and sustaining tumor growth, as this has the potential to reveal therapeutic targets. Stem and progenitor cells have been implicated in the genesis of select lymphoid malignancies. However, the identity of the cells in which mature lymphoid neoplasms are initiated remains unclear. Here, we investigate the origin of peripheral T cell lymphomas using mice in which Snf5, a chromatin remodelling-complex subunit with tumor suppressor activity, could be conditionally inactivated in developing T cells. In this model of mature peripheral T cell lymphomas, the cell of origin was a mature CD44 hi CD122 lo CD8 + T cell that resembled a subset of memory cells that has capacity for self-renewal and robust expansion, features shared with stem cells. Further analysis showed that Snf5 loss led to activation of a Myc-driven signaling network and stem cell transcriptional program. Finally, lymphomagenesis and lymphoma proliferation depended upon TCR signaling, establishing what we believe to be a new paradigm for lymphoid malignancy growth. These findings suggest that the selfrenewal and robust proliferative capacities of memory T cells are associated with vulnerability to oncogenic transformation. Our findings further suggest that agents that impinge upon TCR signaling may represent an effective therapeutic modality for this class of lethal human cancers.

The SWI/SNF chromatin remodeling complex is a master regulator of developmental cell fate decisions, although the key target pathways are poorly characterized. Here, we interrogated the contribution of the SWI/SNF subunit and tumor suppressor SNF5 to the regulation of developmental pathways using conditional mouse and cell culture models. We find that loss of Snf5 phenocopies β-catenin hyperactivation and that SNF5 is essential for regulating Wnt/β-catenin pathway target expression. These data provide insight into chromatin-based mechanisms that underlie developmental regulation and elucidate the emerging theme that mutation of this tumor suppressor complex can activate developmental pathways by uncoupling them from upstream control.

In β-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51–like kinase 1 (Ulk1) gene in β-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 (Atg5) gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in β-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34+cells of β-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating β-thalassemia.