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456 Part V Red Blood Cells

and 26). Activity of GATA1 requires binding to a zinc finger protein derivative, decitabine, inactivate DNA methyltransferases, inducing
cofactor called FOG1 (named for Friend of GATA1). NFE2 recog- γ-globin gene expression and increasing HbF levels in patients with
nizes the DNA sequence motif (T/C) GCT GA (C/G) TCA (T/C). sickle cell anemia. Histone deacetylase inhibitors are being studied as
It is a member of the B-zip class of transcriptional activators. GATA1 agents to increase γ-globin expression.
and NFE2 were originally identified and cloned on the basis of their
interactions with their cognate sequences in the globin genes. Ery-
throid Kruppel-like factor (EKLF, also called KLF1) may be the most Posttranscriptional, Translational, and
specific of the erythroid transcription factors yet discovered. EKLF Posttranslational Mechanisms
interacts specifically with the β-globin gene promoter and may influ-
ence the γ–β switch. Mice homozygous for disruption of the Eklf Processed globin mRNA is exported from the nucleus to the cyto-
gene have lethal β-thalassemia. Alone, GATA1, NFE2, EKLF, and plasm by a mechanism that is not clearly defined. mRNA translation
FOG1 cannot be the sole determination of tissue specificity of the occurs in the cytoplasm (see Fig. 33.7). The triplet codons or mRNA
globin genes. Together, they form a robust transcriptional network are recognized by the anticodons of specific tRNAs that bring acti-
that regulates erythroid genes, including the globin genes. Mutations vated amino acid residues to the nascent polypeptide chains. The
in GATA1 or FOG1 can cause β-thalassemia and thrombocytopenia process of translation, in which an mRNA template directs the syn-
in patients. thesis of protein, is typically divided into three phases: initiation,
The regions of the globin gene clusters with essential regulatory elongation, and termination (see Chapters 1 and 4). Each phase is
sequences and erythroid-specific chromatin remodeling extend far regulated by a variety of protein factors.
beyond the coding sequences of the globin genes. The key regulatory The globin mRNA molecule becomes associated with four to six
elements for the α-globin gene (HS –48, 40, and 33) lies in erythroid- ribosomes, forming the polyribosome. At least 11 eukaryotic transla-
specific DNase I hypersensitive sites about 40 kb upstream from the tion initiation factors interact with the polyribosome. They mediate
α-globin gene. The locus control region (LCR) is critical for high stabilization of a preinitiation complex, binding of the initiator
level expression of the β-globin gene cluster, consisting of five sites methionine tRNA to ribosomal subunits, binding of mRNA to the
that are hypersensitive to DNase I (HS 1–5) (see Chapters 1 and 4). preinitiation complex, stabilization of mRNA binding, recognition
Patients with deletion of the HS –40 sites exhibit α-thalassemia, and of the cap site at the 5′ end of mRNA, and release of initiation factors
patients with deletions of the β-globin LCR develop β-thalassemia; from the preinitiation complex. Several elongation and termination
however, the thalassemia can be a result of changes in chromatin factors have also been defined. Initiation or an early step in the
caused by the large deletion. Similarly, transgenic mice bearing dele- elongation process is the rate-limiting factor.
tions of these critical regulatory regions have severely restricted The first posttranslational step in tetramer formation is the
expressions of the respective globin genes. combination of α-globin and non–α-globin chains to form dimers,
The LCRs contain binding sites for the major erythroid transcrip- an event that appears to depend on the relative charge of each globin
tion factors, including GATA1, EKLF, and NFE2, as well as sites for subunit. The dimers then form tetrameric Hb. Because of charge
transcription factors found more widely distributed in many cell differences among non–α-globin chains, there is a hierarchy or affin-
types. The LCRs loop to interact directly with the promoters of ity of these chains for α-globin chains. The combination of α- and
individual globin genes, resulting in a complex termed the active β-globin chains is most favored followed by a combination of α-, γ-,
chromatin hub. The resulting structure enables high level expression and δ-globin chains. Certain mutant Hbs that have gained or lost a
of globin genes in erythroid cells at the appropriate developmental charge may alter this hierarchic arrangement. This may influence the
stages. Additional elements act as insulators, protecting expressed proportion of variant Hb present, especially when the patient also
genes from gene silencing through the regulation of chromatin inherits an α-thalassemia syndrome, in which the synthesis of
structure. α-globin chains is reduced. The supply of available α-globin chains
BCL11A is a transcriptional repressor that decreases HbF expression is then limited, and non–α-globin chains compete with one another
in adult tissues. Polymorphisms in the BCL11A gene are powerfully to form tetramers with the limiting α-globin chain pool.
associated with HbF levels, including in patients with sickle cell disease. Globin chain biosynthesis and heme synthesis are mutually
Inhibition of Bcl11a increases HbF levels and attenuates the phenotype important. Heme plays a role in the regulation of the initiation
of sickle cell disease in a murine model. The erythroid-specific enhanc- complex. A deficiency of heme (e.g., in iron deficiency) is associated
ers of BCL11A have been identified and are an attractive target for with the accumulation of a repressor of translation initiation factors.
genome editing as a therapeutic strategy for the treatment of sickle cell Translation of β-globin mRNA appears to be initiated more efficiently
disease and β-thalassemia. The transcription factor LRF or ZBTB7A than α-globin mRNA, conferring on the associated anemia some of
is also a powerful silencer of HbF gene expression. When this gene and the features of mild α-thalassemia. This phenomenon occurs because
BCL11A are knocked out in human erythroid cells, the HbF concen- heme deficiency depresses the availability of initiating factors for
tration is more than 90% of the total Hb. which the less efficient α-mRNA must compete with the more effi-
Transcription factors recruit enzymes that remodel chromatin cient β-mRNA.
structure. “Open” chromatin, or euchromatin, generally appears The identification of genetic mutations that cause congenital and
cytogenetically uncondensed and is associated with hyperacetylated acquired forms of anemia provide further insight into the pathways
histones, unmethylated CpG dinucleotides, and active transcription. required for the coordinated production of globin and heme. More
ATRX is a protein that has been implicated in the modulation than half of patients with Diamond-Blackfan anemia, a disorder
of chromatin structure at the α-globin locus. Mutations in the characterized by a severe macrocytic anemia and a paucity of erythroid
ATRX gene, located on the X chromosome, cause a syndrome of progenitor cells, have heterozygous germline mutations in the RPS19
α-thalassemia, severe mental retardation, facial dysmorphism, and gene or other genes encoding ribosomal proteins. Similarly, the
urogenital abnormalities. ATRX, a member of the SNF2 family macrocytic anemia in patients with myelodysplastic syndrome and a
of helicase/ATPases, localizes to pericentromeric heterochromatin deletion of chromosome 5q is caused by heterozygous deletion of
during interphase and mitosis and contains a plant homeodomain- another ribosomal protein gene, RPS14. Haploinsufficiency for these
like domain that is found in chromatin-associated proteins. Cells ribosomal protein genes activates the p53 pathway, leading to cell
with a mutated ATRX gene have altered patterns of DNA methyla- cycle arrest and apoptosis selectively in the erythroid progenitor cells.
tion. The ATRX protein therefore exemplifies the connections among Refractory anemia with ring sideroblasts (RARS) is a subtype of
DNA methylation, chromatin remodeling, and expression of the myelodysplastic syndrome characterized by iron-loaded mitochondria
α-globin genes. evident on Prussian blue staining. In the majority of RARS cases,
Chromatin structure can be manipulated pharmacologically somatic mutations are present in the SF3B1 gene, encoding a core
through the influence of drugs on methylation and histone acetyla- member of the RNA splicing machinery. The precise targets of SF3B1
tion. Cytidine analogs such as 5-azacytidine and its less toxic have not been identified.