Reconstitution of Human β-Globin Locus Control Region Hypersensitive Sites in the Absence of Chromatin Assembly

Leach, Kelly M.; Nightingale, Karl P.; Igarashi, Kazuhiko; Levings, Padraic P.; Engel, James Douglas; Becker, Peter B.; Bungert, Jörg

doi:10.1128/mcb.21.8.2629-2640.2001

Cited by 53 publications

(55 citation statements)

References 46 publications

(66 reference statements)

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“…1B can be explained by a mechanism in which Pol II is recruited directly to the HSs, but it is instructive to consider other mechanisms. As human HS2 and HS3 function as promoters (31,50), a small fraction of Pol II, undetectable by ChIP, might engage in extragenic transcription. Alternatively, Pol II might be recruited upstream of the HSs, and the accumulation of Pol II at the HSs might reflect strong pausing of elongating Pol II at the HSs.…”

Section: Resultsmentioning

confidence: 99%

“…An alternate model for Pol II function at the ␤-globin LCR is suggested by the presence of extragenic transcripts at the ␤-globin locus (2). Pol II transcription through the LCR, originating from an upstream ERV9 long terminal repeat (36,46) and from HS2 and HS3 in human erythroid cells (31), has been proposed to mediate chromatin modification (16), potentially by mobilizing Pol II-associated chromatin-modifying complexes (64,65). Extragenic transcripts have also been detected at the major histocompatibility complex class II locus (40) and at the Drosophila bithorax complex (3).…”

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confidence: 99%

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Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain

Johnson

Grass

Park

et al. 2003

Molecular and Cellular Biology

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RNA polymerase II (Pol II) can associate with regulatory elements far from promoters. For the murine ␤-globin locus, Pol II binds the ␤-globin locus control region (LCR) far upstream of the ␤-globin promoters, independent of recruitment to and activation of the ␤major promoter. We describe here an analysis of where Pol II resides within the LCR, how it is recruited to the LCR, and the functional consequences of recruitment. High-resolution analysis of the distribution of Pol II revealed that Pol II binding within the LCR is restricted to the hypersensitive sites. Blocking elongation eliminated the synthesis of genic and extragenic transcripts and eliminated Pol II from the ␤major open reading frame. However, the elongation blockade did not redistribute Pol II at the hypersensitive sites, suggesting that Pol II is recruited to these sites. The distribution of Pol II did not strictly correlate with the distributions of histone acetylation and methylation. As Pol II associates with histone-modifying enzymes, Pol II tracking might be critical for establishing and maintaining broad histone modification patterns. However, blocking elongation did not disrupt the histone modification pattern of the ␤-globin locus, indicating that Pol II tracking is not required to maintain the pattern.Although well-defined basal transcription factors are required for transcription initiation, the assembly of these factors on promoters is highly regulated through transcription factors bound at promoters and at distal enhancers and locus control regions (LCRs) (7, 23). Transcription factors recruit basal factors through protein-protein interactions (47) and indirectly by recruiting chromatin-modifying coactivators, which increase promoter accessibility (5, 32, 60). While it is easy to envision how promoter-bound factors activate transcription, the mechanisms controlling transcription over a long distance on a chromosome are poorly understood. Evidence strongly supports a looping mechanism, in which chromatin structure juxtaposes distal regulatory elements with the gene, thereby allowing contact between factors bound at distant sites (6).Looping has been implicated in the transcriptional control of the murine ␤-globin genes (8, 56). The ␤-globin locus consists of four genes arrayed in the order in which they are expressed developmentally (Fig. 1A). High-level transcription of the ␤-globin genes requires upstream DNase I-hypersensitive sites (HS1 to HS4) (12, 59) referred to as the LCR (Fig. 1A) (4,10,11,17). As shown for erythroid cells from murine fetal liver, the LCR is positioned close to the active adult ␤-globin genes but not the inactive embryonic genes (8, 56), despite the fact that the embryonic genes are closer to the LCR linearly on the chromosome. However, determinants for establishing looping have not been defined, nor it is known how looping activates transcription, even though factors that bind the LCR and promoter in vivo are known (14,25,33,52). Looping also occurs within the HNF4␣ locus, and a tracking mechanism was propos...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain

Johnson

Grass

Park

et al. 2003

Molecular and Cellular Biology

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“…LCRs may recruit transcription factors and components of the transcription preinitiation complex, including RNA polymerase II, that are subsequently transferred to the promoter region (25,32,47). Our finding that Pol II is bound to HS3 in the 5Ј HS cluster and the promoter region in WT mast cells and BMMC isolated from W sh/sh and BAC200-Kit-GFP transgenic mice, but not in the promoter of the GFP-negative BMMC isolated from W sh/sh , BAC30-Kit-GFP, and BAC200-⌬5ЈHS-Kit-GFP mice, is consistent with such a mechanism.…”

Section: Discussionmentioning

confidence: 99%

A Distant Upstream Locus Control Region Is Critical for Expression of the Kit Receptor Gene in Mast Cells

Berrozpe¹,

Agosti²,

Tucker³

et al. 2006

Molecular and Cellular Biology

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The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5 HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5 upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5 HS region were associated with Kit expression. Therefore, the 5 upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.Differential control of gene expression during embryonic development and in the adult organism is mediated by the interaction of the transcription machinery with cis regulatory elements located at the promoter, upstream, or in intronic sequences of a gene. The mechanism by which tissue-specific gene expression is achieved involves the action of transcription factors together with changes in chromatin structure. Chromatin structure is a major determinant of gene expression, and mechanisms of chromatin remodeling are critical components of its regulation. Whereas SWI/SNF-like chromatin-remodeling ATPases disrupt histone-DNA interactions, covalent Nterminal histone modifications, including acetylation, methylation, and phosphorylation, also have important roles in the remodeling of chromatin structure and the regulation of transcription (36). In this way histone modification patterns have been proposed to constitute a histone "code" which specifies downstream functions (45). Hyperacetylation and hypoacetylation of histones H3 and H4 correlate with open/accessible or closed/inaccessible chromatin structures, respectively, and transcriptional activation or repression (22, 31). Thus, the characterization of histone modification patterns has become an important tool in studies of gene expression.The Kit receptor tyrosine kinase (RTK) functions in distinct cell populations during embryonic development and in the postnatal animal (5, 6). During gametogenesis, Kit is expressed in primordial germ cells as they migrate from the allantois to the genital ridge (10, 35). Subsequently, during postnatal gametogenesis, Kit is expressed in spermatogonia and oocytes and in endocrine Leydig and thecal cells (2). In hematopoiesis during embryogenesis and in postnatal animals, Kit is expressed in hematopoietic stem cells and lineage progenitors, as well as in mast cells and eosinophils (5, 18, 48). In melanogenesis, Kit is expressed in migrating melanoblasts during embryonic development, in differentiated melanocytes in hair follicles, and in the gastrointestinal tract in ...

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“…Previous studies have shown that Pol II interacts with LCR HS2 and with the adult β-globin gene promoter and that the binding increases on induction of MEL cell differentiation (26)(27)(28)(29). As shown in Fig.…”

Section: Resultsmentioning

confidence: 89%

Neutralizing the function of a β-globin–associated cis -regulatory DNA element using an artificial zinc finger DNA-binding domain

Barrow

Masannat

Bungert

2012

Proc. Natl. Acad. Sci. U.S.A.

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Gene expression is primarily regulated by cis-regulatory DNA elements and trans-interacting proteins. Transcription factors bind in a DNA sequence-specific manner and recruit activities that modulate the association and activity of transcription complexes at specific genes. Often, transcription factors belong to families of related proteins that interact with similar DNA sequences. Furthermore, genes are regulated by multiple, sometimes redundant, cis-regulatory elements. Thus, the analysis of the role of a specific DNA regulatory sequence and the interacting proteins in the context of intact cells is challenging. In this study, we designed and functionally characterized an artificial DNA-binding domain that neutralizes the function of a cis-regulatory DNA element associated with adult β-globin gene expression. The zinc finger DNA-binding domain (ZF-DBD), comprising six ZFs, interacted specifically with a CACCC site located 90 bp upstream of the transcription start site (-90 β-ZF-DBD), which is normally occupied by KLF1, a major regulator of adult β-globin gene expression. Stable expression of the -90 β-ZF-DBD in mouse erythroleukemia cells reduced the binding of KLF1 with the β-globin gene, but not with locus control region element HS2, and led to reduced transcription. Transient transgenic embryos expressing the -90 β-ZF-DBD developed normally but revealed reduced expression of the adult β-globin gene. These results demonstrate that artificial DNA-binding proteins lacking effector domains are useful tools for studying and modulating the function of cis-regulatory DNA elements.artificial transcription factor | red cell | gene regulation

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Reconstitution of Human β-Globin Locus Control Region Hypersensitive Sites in the Absence of Chromatin Assembly

Cited by 53 publications

References 46 publications

Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain

Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain

A Distant Upstream Locus Control Region Is Critical for Expression of the Kit Receptor Gene in Mast Cells

Neutralizing the function of a β-globin–associated cis -regulatory DNA element using an artificial zinc finger DNA-binding domain

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