A newly discovered human α-globin gene

Abstract

IntroductionThe globin genes and their products have been intensively investigated for the past 50 years. Those studies led to the description of structural and regulatory elements that are useful for the recognition and comparison of hundreds of globin gene family members. The divergence of ancestral ␣-and ␤-globin genes is estimated to have occurred 500 million years ago. Those genes subsequently evolved and were modified by a variety of genetic processes, including duplication events. 1 In humans, the ␣-globin gene family resides on chromosome 16p13.3 and is composed of a cluster of 3 genes (2-␣2-␣1) with protein products that bind heme and assemble into hemoglobin. Transcription of the 2 gene is silenced during fetal life, and the 2 ␣ genes (␣2 and ␣1) are expressed in a balanced fashion for the remainder of ontogeny. In addition to those 3 genes, concerted efforts in the 1980s led to the discovery of other ␣-like sequences. The downstream region of the ␣ locus contains an unusual gene named -globin that generates no detected globin protein in humans. 2,3 -globin gene transcription is regulated, and the transcripts contain no obvious defects to explain the lack of detectable protein in erythroid tissues. 2-5 Three pseudo-globin genes (pseudo-1, 6 pseudo-␣2, 7 and pseudo-␣1 8 ) were also identified in the ␣-globin locus.During the past 2 years, investigators have begun a transition toward postgenomic approaches to basic and clinical research. Hypotheses are now generated with the knowledge of whole genomic DNA sequences, 9 full-length cDNA collections, 10 and millions of expressed sequence tags (ESTs) 11 from humans and other species. Comparisons of DNA and RNA sequences with advanced bioinformatics analyses 12 have become essential. Hematology is ideally suited for this type of genome-based research due to the ease with which purified populations of hematopoietic cells are isolated. We hypothesized that human reticulocytes contain sufficient mRNA from the terminal stages of differentiation for the study of globin gene expression patterns in high throughput. Levels of globin mRNA detection and differences in globin gene transcription between cord and adult blood were studied to determine the potential of this approach for clinical assessments of hemoglobinopathies and hemoglobin switching. Using oligonucleotide arrays, significantly different globin transcription patterns were found in cord and adult blood samples. Evidence for transcription of the major globin genes was clearly demonstrated. Surprisingly, we also identified transcription from the genomic region previously thought to encode the pseudo-␣2 gene. The source of that transcription is characterized in this report as a previously unrecognized globin gene. Materials and methods Preparation of reticulocyte RNABlood was collected from healthy adult donors and from placental umbilical cords. All cells were collected according to approved guidelines regarding human subjects. Blood samples were centrifuged, and plasma and buffy coat layers were removed. P...

IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

“…Quantitative real-time PCR amplification was carried out in a 7900HT Fast Real-Time PCR System or 7500 Real Time PCR System (Thermo Fisher Scientific) using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) following manufacturer's instructions. RT-qPCR assays and conditions were performed as previously described (11,29,55,56). The following commercially available Assay-on-Demand gene expression products (Thermo Fisher Scientific) were used: IGF2BP1 (Hs00977566_m1), IGF2BP2 (Hs00538956_m1), IGF2BP3 (Hs01122560_g1), CA1 (Hs01100176_m1), GCNT2 (Hs00377334_m1), BCL11A (Hs00256254_m1), HMGA2 (Hs00971724_m1), ZBTB7A (Hs00792219_m1), KLF1 (Hs00610592_m1), SOX6 (Hs00264525_m1), c-MYC (Hs00153408_m1), IGF2 (Hs01005963_m1), and Beta-Actin (Hs99999903_m1).…”

Primitive and definitive erythropoiesis in the yolk sac: a birds eye view

“…pi is orthologous to human zeta/pseudo-zeta, alphaD to human mu (also called pseudo-alpha2 or alphaD) and alphaA to human pseudo-alpha1/ alpha2/alpha1/theta. Individual beta hemoglobin genes in chickens and mammals arose by independent duplications and do not form orthologous groups, although their ancestral beta genes before duplication are considered orthologous (Alev et al, 2009;Cooper et al, 2006;Goh et al, 2005;Patel et al, 2008). Primitive erythrocytes in chickens have high pi, rho and epsilon hemoglobin proteins, and definitive erythrocytes have high alphaD, alphaA and betaA.…”

Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming

“…Quantitative PCR assays were performed with primers, probes, and PCR conditions described previously for ␥-, ␤-, -, and ␣-globin. 17 In addition, amplifications for ⑀-, ␦-, -, and -globin transcripts were performed using Assays-on-Demand Gene Expression Products (Applied Biosystems) according to the manufacturer's instructions. Individual globin copy numbers were calculated by comparison with standard curves generated from a plasmid DNA encoding each globin template.…”