Briefings in Functional Genomics and Proteomics

Briefings in Functional Genomics and Proteomics Advance Access originally published online on February 22, 2006
Briefings in Functional Genomics and Proteomics 2006 5(1):62-65; doi:10.1093/bfgp/ell001

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Special Issues Papers

Multi-protein complexes at the ß-globin locus

Milind C. Mahajan and Sherman M. Weissman

Corresponding author. Sherman M. Weissman, Department of Genetics, The Anlyan Center, Yale University School of Medicine, 300 Cedar St., New Haven, CT 06510, USA. Tel: +1 203 737 2281/82; Fax: +1 203 737 2286. E-mail: Sherman.weissman{at}yale.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 
Biochemical analysis of the ß-globin gene function has led to the identification of several multi-protein complexes at the locus control region (LCR), insulator and promoters. This review briefly summarizes these multi-protein complexes and discusses their contribution towards the regulation of the ß-globin gene expression.

Keywords: ß-globin, LCR, SWI/SNF, MeCP1, CTCF, hnRNP C1/C2, GATA-1, NF-E2, EKLF

	INTRODUCTION

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 
The 73kb ß-globin locus on human chromosome 11 has been extensively studied over the years as a model system for the developmental regulation of gene expression in a tissue-specific manner, molecular mechanisms of enhancer–promoter interactions, properties of insulator and locus control region (LCR), and a target for gene therapy as a cure for various haemoglobinopathies. These studies have led to the identification of several tissue-restricted transcription factors such as GATA-1, EKLF, NF-E2, TAL-1, Bach1 and Bach2 that play an important role in erythroid differentiation as well as the regulation of ß-globin gene expression. Increasingly, it is becoming clear that these factors do not act independently but as part of multi-protein complexes. Such complexes might only be assembled on DNA but often pre-exist as heteromeric complexes in the nucleoplasm. The characterization of these assemblages and their role in globin gene expression is a relatively new and undeveloped aspect of the study of these well-known genes. We summarize here some of the recent studies that are uncovering the occurrence and nature of such complexes (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1: A schematic diagram showing the positions of the DNA-binding sites for the high molecular weight multi-protein complexes on the ß-globin locus.

 

	COMPLEXES INCLUDING GATA-1

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 
GATA-1 is one of the most extensively studied cell-type-specific transcription factors. It has been demonstrated to associate with a number of other factors including FOG-1, TAL-1, CBP/P300, PU.1, Sp1 and Gfi1. Recent studies indicate that GATA-1 exists in several separate complexes, respectively containing histone deacetylating, methylated DNA binding and chromatin remodelling NuRD/MeCP1 complex, chromatin remodelling ACF/WRCF complex, DNA repair proteins, DNA topoisomerases and transcription factors such as TAL-1, FOG-1, Gfi-1b and Ldb1 [1, 2]. Among these transcription factors, FOG-1 is necessary to bridge the association between GATA-1 and MeCP1 complex. Interestingly, GATA-1 is known to repress transcription of some genes such as GATA-2, a factor expressed during earlier stages of haematopoietic differentiation. This repression occurs in the same cells in which GATA-1 and FOG-1 act as activators for other promoters. Elegant evidence is presented to show that the repressive activity of GATA-1 is mediated through its recruitment of MeCP1 on the GATA-2 promoter [1, 2]. At other promoters GATA-1 binding may be associated with gene repression, perhaps mediated by a complex between GATA-1 and Gfi-1b. On the other hand, at least at the EKLF promoter, GATA-1 activation may be associated with a complex including TAL-1. The precise nature of the sequences and factors that determine which GATA-1 interactions occur at a particular promoter are of general interest and largely remain to be determined. Also, it is not presently clear whether and which preformed GATA-1 complexes might occur in the absence of specific DNA sequences and what processes lead to assembly of specific GATA-1 complexes. GATA-1 binds to several sites at the ß-globin locus [3, 4]. It remains to be seen which of these GATA-1 complexes interact with the known GATA-1 binding sites on the ß-globin locus and its significance in the developmental regulation of the ß-like globin gene transcription and chromatin structure.

	COMPLEXES INCLUDING NF-E2 OR BACH1

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 
p45NF-E2 is a relatively erythroid-specific member of a group of related transcription factors [5–7]. These factors function as heterodimers with Maf subunits to bind to the enhancer region of the ß-globin LCR. Bach1 is a second transcription factor that interacts with Maf subunits and binds to the same sites as does NF-E2 within the LCR [8, 9]. Bach1 acts as a negative regulator of transcription, and the alternate binding of NF-E2 versus Bach1 has been proposed as a part of a regulatory mechanism for globin production [10]. Bach1 and p45NF-E2 occur in a large protein complex that can be partially purified by column chromatography and size-fractionation, but the nature of this complex and associated proteins remains to be determined [10], Mahajan et al. (unpublished results) (Figure 1).

	CTCF-ASSOCIATED COMPLEX AT THE INSULATOR

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 
Insulators are the DNA elements that block enhancer–promoter interaction and the spread of the heterochromatin [11]. Insulator DNA elements reside at the HS4 region of the chicken ß-globin LCR and HS5 of the human ß-globin LCR. A zinc finger containing DNA-binding protein called CTCF binds to the insulator element [12]. Conventional biochemical purification of the CTCF and its associated proteins suggest its association with nucleolar nucleophosmin and PARP, 40S ribosomal subunits, topoisomerase II, lamin A/C, histones H2A and H2Az, and template-activating factor Taf-1/set [13]. The in vivo association of these CTCF-binding proteins at the insulator sequence and their significance for the insulator activity is yet to be described.

	COMPLEXES INCLUDING CHROMATIN REMODELLING FACTORS

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 
Chromatin remodelling and nucleosome movement may be catalysed by one of several DNA-dependant ATPases, usually in association with a multi-protein complex. Of these ATPases, BRG1 is perhaps the most abundant and extensively studied. BRG1 is found in a major protein assemblage known as the SWI/SNF complex [14–16]. This SWI/SNF complex does not itself have sequence-specific DNA-binding properties, but may be attracted to particular sequences by interaction with other transcription factors. In particular, in the case of globin, SWI/SNF is recruited on the ß-globin gene promoter by virtue of its association with the EKLF-transcription factor necessary for efficient ß-globin production [17, 18].

Unlike the associations described for GATA-1 and EKLF, the study of chromatin-remodelling complexes associated with the ß-globin cluster has sometimes begun by biochemical purification of a multi-component complex as a DNA-binding activity. In one such case, Arthur Bank and associates have extensively purified an Ikaros-containing SWI/SNF protein complex called PYR complex that binds to the pyrimidine rich sequence 5' upstream of the -globin gene [19]. A delayed fetal-to-adult globin switching was observed in transgenic mice with deletion of the PYR complex binding sequence as well as in the Ikaros null mice [19, 20]. Further purification studies of the complexes binding to this pyrimidine-rich sequence have shown that Ikaros is also associated with the NuRD complex and recruits both SWI/SNF as well as NuRD complexes to the same sequence in vitro [21].

Recently a high molecular weight complex containing SWI/SNF and MeCP1 has been purified to near homogeneity from K562 erythroleukaemic cells [22]. This complex was shown to bind with sequence specificity to the core enhancer region of the ß-globin LCR hypersensitive site 2. In vivo chromatin-immunoprecipitation studies showed that BRG1, components of the MeCP1 complex, and hnRNPC were associated with the LCR HS2 and promoter regions of the two -globin genes and the ß-globin gene (Figure 1). The enrichment of ChIP–PCR products at HS2 and ß-like gene promoters were similar, suggesting that the LARC may be the major complex detectable by this method in vivo. The results are curious in that K562 cells produce -globin mRNA, small amounts of embryonic globin mRNA, but virtually no ß-globin mRNA. This indicates that, as a minimum, LARC can bind to promoters before transcription initiates.

HLTF is another SWI2/SNF2-related DNA-dependent ATPase whose sequence specifically associates with the –115 to –140 segment of the ß-globin promoter sequences in vitro [23]. On a gel filtration column, it co-elutes with the components of SWI/SNF complex as a 1.5 MDa protein complex (Mahajan et al., unpublished results). Other constituents of this complex have not yet been identified. Interestingly, overexpression of HLTF in K562 cells resulted in a substantial increase in the amount of detectable ß-globin mRNA. This occurred even though the transcription factor EKLF, thought to be necessary for normal levels of ß-globin mRNA production, was absent from the cells. No other factor has yet been described that produces a similar effect and further studies of the possible roles of HLTF in globin gene expression are clearly warranted.

There is a general question underlying the study of these various multi-protein complexes—to what extent might the conditions of isolation including inadvertent limited proteolysis as well as chromatographic separation conditions lead to dissociation of sub-complexes that are in even larger assemblages in vivo. When K562 nuclei are extracted in the presence of large amounts of protease inhibitors and then fractionated by heparin agarose chromatography and sized on gel filtration columns, a large fraction of the factors associated with multi-protein complexes at the LCR and ß-globin promoter actually emerge close to the void volume of the Superose-6 sizing column, indicating a molecular mass much in excess of 2 MDa (Mahajan et al., unpublished data). Chromatin configuration studies indicate that the LCR and promoters may be associated in a hub and presumably this association is mediated by interactions between proteins or proteins and RNA. There are other reports of the isolation of such giant complexes involving chromatin-remodelling subunits along with other types of proteins [24]. It is tempting to speculate that several of the multi-protein complexes exist separately in vivo and that a role of LCR elements is to act as a catalyst or scaffold for assembly of giant complexes from the pre-existing very large sub-complexes.

Key Points ß-Globin locus binding transcription factors, such as GATA-1, NF-E2, EKLF and HLTF, are associated with large multi-protein complexes. The chromatin-remodelling complexes are associated with LCR as well as ß-like globin promoters and their upstream sequences. The chromatin-remodelling complexes associated with the ß-globin gene promoter and upstream of the -globin gene promoter are recruited through the DNA-binding transcription factors. In case of LCR-associated chromatin-remodelling complex, the hnRNP C1/C2 provides the sequence-specific DNA-binding property. The insulator sequence at the 5' end of the LCR is associated with the CTCF-containing protein complex.

 

	FOOTNOTES

 
Milind C. Mahajan is an Associate Research Scientist in Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.

Sherman M. Weissman is a Sterling Professor of Genetics and Medicine at Yale University School of Medicine. He is the Co-Director of the Molecular Oncology and Development Program at the Yale Cancer Center, and Co-Director of Yale's Center for Excellence in Genomic Science.

	References

TOP ABSTRACT INTRODUCTION COMPLEXES INCLUDING GATA-1 COMPLEXES INCLUDING NF-E2 OR... CTCF-ASSOCIATED COMPLEX AT THE... COMPLEXES INCLUDING CHROMATIN... References

 

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