Publications by authors named "Jacqueline Sloane-Stanley"

19 Publications

  • Page 1 of 1

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Blood Adv 2021 08;5(15):3002-3015

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.
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http://dx.doi.org/10.1182/bloodadvances.2021004609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361462PMC
August 2021

Reactivation of a developmentally silenced embryonic globin gene.

Nat Commun 2021 07 21;12(1):4439. Epub 2021 Jul 21.

MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.
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http://dx.doi.org/10.1038/s41467-021-24402-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295333PMC
July 2021

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.

Nat Cell Biol 2017 Aug 24;19(8):952-961. Epub 2017 Jul 24.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK.

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.
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http://dx.doi.org/10.1038/ncb3573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540176PMC
August 2017

Enhancer deletion generates cellular phenotypic diversity due to bimodal gene expression.

Blood Cells Mol Dis 2017 05 1;64:10-12. Epub 2017 Mar 1.

The Roslin Institute, Developmental Biology Division, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK. Electronic address:

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http://dx.doi.org/10.1016/j.bcmd.2017.02.006DOI Listing
May 2017

Genetic dissection of the α-globin super-enhancer in vivo.

Nat Genet 2016 08 4;48(8):895-903. Epub 2016 Jul 4.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford, UK.

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5058437PMC
http://dx.doi.org/10.1038/ng.3605DOI Listing
August 2016

High-resolution analysis of cis-acting regulatory networks at the α-globin locus.

Philos Trans R Soc Lond B Biol Sci 2013 6;368(1620):20120361. Epub 2013 May 6.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, UK.

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.
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http://dx.doi.org/10.1098/rstb.2012.0361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682726PMC
December 2013

Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

Hum Mutat 2013 Aug 18;34(8):1140-8. Epub 2013 Jun 18.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.
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http://dx.doi.org/10.1002/humu.22343DOI Listing
August 2013

Nprl3 is required for normal development of the cardiovascular system.

Mamm Genome 2012 Aug 27;23(7-8):404-15. Epub 2012 Apr 27.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK.

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.
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http://dx.doi.org/10.1007/s00335-012-9398-yDOI Listing
August 2012

Intragenic enhancers act as alternative promoters.

Mol Cell 2012 Feb 19;45(4):447-58. Epub 2012 Jan 19.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK.

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.
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http://dx.doi.org/10.1016/j.molcel.2011.12.021DOI Listing
February 2012

An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

EMBO J 2012 Jan 4;31(2):317-29. Epub 2011 Nov 4.

MRC Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.
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http://dx.doi.org/10.1038/emboj.2011.399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261560PMC
January 2012

Polycomb eviction as a new distant enhancer function.

Genes Dev 2011 Aug;25(15):1583-8

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes.
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http://dx.doi.org/10.1101/gad.16985411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182025PMC
August 2011

Generation of bivalent chromatin domains during cell fate decisions.

Epigenetics Chromatin 2011 Jun 6;4(1). Epub 2011 Jun 6.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Background: In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined.

Results: Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3.

Conclusions: While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.
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http://dx.doi.org/10.1186/1756-8935-4-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131236PMC
June 2011

Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts.

Blood 2011 Jun 1;117(25):6928-38. Epub 2011 Mar 1.

Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.
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http://dx.doi.org/10.1182/blood-2010-09-308478DOI Listing
June 2011

Chromosome looping at the human alpha-globin locus is mediated via the major upstream regulatory element (HS -40).

Blood 2009 Nov 20;114(19):4253-60. Epub 2009 Aug 20.

Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute for Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS United Kingdom.

Previous studies in the mouse have shown that high levels of alpha-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human alpha cluster and their interactions with the alpha-globin promoter. All of these elements interact with the alpha-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human alpha globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS -40) results in loss of all the interactions and alpha-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human alpha-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.
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http://dx.doi.org/10.1182/blood-2009-03-213439DOI Listing
November 2009

A large deletion in the human alpha-globin cluster caused by a replication error is associated with an unexpectedly mild phenotype.

Hum Mol Genet 2008 Oct 15;17(19):3084-93. Epub 2008 Jul 15.

Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK.

We have characterized a newly identified 16.6 kb deletion which removes a significant proportion of the human alpha-globin cluster including the psizeta1, alpha(D), psialpha1 and alpha2-globin genes but leaves the duplicated alpha1 gene intact. This complicated rearrangement results from a combination of slippage and strand switching at sites of microhomology during replication. Functional analysis shows that expression of the remaining alpha1 gene is increased, rather than down-regulated by this deletion. This could be related to its proximity to the remote upstream alpha-globin regulatory elements or reduced competition for these elements in the absence of the dominant alpha2-globin gene. The finding of a very mild phenotype associated with such an extensive deletion in the alpha-globin cluster implies that much of the DNA removed by the deletion is likely to be functionally unimportant. These findings suggest that other than the upstream regulatory elements and promoter proximal elements there are unlikely to be additional positive cis-acting sequences in the alpha-globin cluster.
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http://dx.doi.org/10.1093/hmg/ddn205DOI Listing
October 2008

Tissue-specific histone modification and transcription factor binding in alpha globin gene expression.

Blood 2007 Dec 22;110(13):4503-10. Epub 2007 Aug 22.

Medical Research Council, Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, UK.

To address the mechanism by which the human globin genes are activated during erythropoiesis, we have used a tiled microarray to analyze the pattern of transcription factor binding and associated histone modifications across the telomeric region of human chromosome 16 in primary erythroid and nonerythroid cells. This 220-kb region includes the alpha globin genes and 9 widely expressed genes flanking the alpha globin locus. This un-biased, comprehensive analysis of transcription factor binding and histone modifications (acetylation and methylation) described here not only identified all known cis-acting regulatory elements in the human alpha globin cluster but also demonstrated that there are no additional erythroid-specific regulatory elements in the 220-kb region tested. In addition, the pattern of histone modification distinguished promoter elements from potential enhancer elements across this region. Finally, comparison of the human and mouse orthologous regions in a unique mouse model, with both regions coexpressed in the same animal, showed significant differences that may explain how these 2 clusters are regulated differently in vivo.
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http://dx.doi.org/10.1182/blood-2007-06-097964DOI Listing
December 2007

Defining the cause of skewed X-chromosome inactivation in X-linked mental retardation by use of a mouse model.

Am J Hum Genet 2007 Jun 25;80(6):1138-49. Epub 2007 Apr 25.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.

Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.
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http://dx.doi.org/10.1086/518369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867101PMC
June 2007

Long-range chromosomal interactions regulate the timing of the transition between poised and active gene expression.

EMBO J 2007 Apr 22;26(8):2041-51. Epub 2007 Mar 22.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK.

To understand how mammalian genes are regulated from their natural chromosomal environment, we have analysed the molecular events occurring throughout a 150 kb chromatin segment containing the alpha globin gene locus as it changes from a poised, silent state in erythroid progenitors, to the fully activated state in late, erythroid cells. Active transcription requires the late recruitment of general transcription factors, mediator and Pol II not only to the promoter but also to its remote regulatory elements. Natural mutants of the alpha cluster show that whereas recruitment of the pre-initiation complex to the upstream elements occurs independently, recruitment to the promoter is largely dependent on the regulatory elements. An improved, quantitative chromosome conformation capture analysis demonstrates that this recruitment is associated with a conformational change, in vivo, apposing the promoter with its remote regulators, consistent with a chromosome looping mechanism. These findings point to a general mechanism by which a gene can be held in a poised state until the appropriate stage for expression, coordinating the level and timing of gene expression during terminal differentiation.
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http://dx.doi.org/10.1038/sj.emboj.7601654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852780PMC
April 2007

Deletion of the mouse alpha-globin regulatory element (HS -26) has an unexpectedly mild phenotype.

Blood 2002 Nov 5;100(10):3450-6. Epub 2002 Jul 5.

Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom.

Natural deletions of the region upstream of the human alpha-globin gene cluster, together with expression studies in cell lines and transgenic mice, identified a single element (HS -40) as necessary and perhaps sufficient for high-level expression of the alpha-globin genes. A similar element occupies the corresponding position upstream of the mouse (m) alpha-globin genes (mHS -26) and was thought to have similar functional properties. We knocked out mHS -26 by homologous recombination and observed the surprising result that instead of the expected severe alpha-thalassemia phenotype, the mice had a mild disease. Transcription levels of the mouse genes were reduced by about 50%, but homozygotes were healthy, with normal hemoglobin levels and only mild decreases in mean corpuscular volume and mean corpuscular hemoglobin. These results may indicate differences in the regulation of the alpha-globin clusters in mice and humans or that additional cis-acting elements remain to be characterized in one or both clusters.
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http://dx.doi.org/10.1182/blood-2002-05-1409DOI Listing
November 2002
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