Partial repression of human γ-globin genes by LCR element HS3 when linked to β-globin genes and LCR element HS2 in MEL cells

“…Subsequent studies identified this same sequence as an enhancer element, which was able to increase transcription levels of a reporter gene after transient transfection into both erythroid and nonerythroid cell lines (4). However, later studies examining various fragments of the locus reported that this same sequence possessed silencer activity (6) or indeed had no effect at all (19). Although yet a fifth analysis implicated the region in matrix or chromosome scaffold attachment (7), most recently a study examining the activity of various LCR͞A␥-globin constructs in the presence or absence of the A␥3ЈE concluded that it functions as a chromatin insulator, which, in collaboration with the LCR, confers position-independent expression to the ␥-globin genes in transgenic mice (8,20).…”

Section: Discussionmentioning

confidence: 99%

The Aγ-globin 3′ element provides no unique function(s) for human β-globin locus gene regulation

Liu

Tanimoto²,

Bungert³

et al. 1998

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

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The human ␤-globin locus is activated transcriptionally by a complex series of events that culminate in appropriate temporal and tissue-specific control over five separate genes during embryonic and early postnatal development. One cis-regulatory element in the locus, originally identified as an enhancer 3 to the A␥-globin gene, more recently has been suggested to harbor alternative or additional properties, including stage-specific silencer, insulator, nuclear matrix, or chromosome scaffold attachment activities. We have re-evaluated the activity during erythropoiesis that is conferred by this element by deleting it from a yeast artificial chromosome (YAC) containing the entire human ␤-globin locus and then assaying for the expression of each gene at each developmental stage after incorporation of the mutant YAC into the mouse germline. The data show that loss of the A␥-globin 3 element confers no phenotype in six independent lines of intact YAC mutant transgenic mice, thus demonstrating (minimally) that any activities attributable to this element are fully compensated by other DNA sequences within the ␤-globin locus.At least 17 discrete transcriptional control elements lie within the human ␤-globin gene locus. These include the five DNaseI hypersensitive sites lying from 6 to 24 kbp 5Ј to the -globin gene [the locus control region (LCR) (1-3)], the promoters of each of five separate genes, and distinct modulatory elements that lie either within, or in relatively close proximity to, one or another of the genes.Among these gene-proximal regulatory elements, one of the earliest to be identified was an enhancer element discovered over a decade ago in transient transfection experiments (4). This sequence lies 410 bp 3Ј to the A␥-globin gene and is confined within boundaries originally described by a 757-bp EcoRI͞HindIII fragment. With passing time, however, the bona fide regulatory activity conferred by this element became more controversial because subsequent studies in transgenic mice implicated this same element in the stage-specific and autonomous silencing of the A␥-globin gene in adult stage erythroid cells (5, 6). Subsequently, in vitro studies implicated the same element as a chromatin structural motif that could participate in tethering the locus to chromosome scaffolds or to the nuclear matrix (7). More recently, it was shown that this sequence, in collaboration with the LCR, is able to shelter ␥-globin transgenes from variegation effects caused by the neighboring chromatin into which they randomly integrate, suggesting that this combination of cis-elements can provide insulation from the repressive effects of surrounding heterochromatin (8). However, even the most current analyses demonstrate that the activity due to this sequence is still controversial and unresolved (9). These multiple observations and seemingly disparate conclusions then provoke several questions: Is this A␥-globin 3Ј element (referred to hereafter as the A␥3ЈE) an enhancer, a silencer, or a sequence required for appropriate c...

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“…While most LCRs are associated with enhancer activity, LCRs can also be repressive. When γ-globin and β-globin were cloned together with their LCR, the LCR element HS3 promoted β-globin while repressing γ-globin [147]. This data supports the notion that an LCR can act as an enhancer for one gene but act as a repressor on another.…”

Section: Locus Control Regions (Lcrs)supporting

confidence: 73%

“…The most well characterised LCR is that regulating the human -globin locus [147][148][149]. This LCR consists of five HS sites that are located 6-20kb upstream of the five β-type globin genes on chromosome 11 [150].…”

Section: Locus Control Regions (Lcrs)mentioning

confidence: 99%

Identifying long-range control elements of the BRCA1 promoter

Lee¹

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BRCA1 is a tumour suppressor protein involved in many important pathways such as DNA repair, cell cycle regulation, apoptosis, spindle formation and telomere regulation. BRCA1 germline mutations confer the highest risk to breast cancer. Furthermore, reduced levels of BRCA1 are also observed in many sporadic breast cancers. Despite the obvious importance of BRCA1, genetic screening is usually restricted to BRCA1 exons and intronexon boundaries. The possibility that mutations exist outside these regions has not been fully investigated and may be responsible for breast cancer predisposition in a subset of . However, identifying regulatory elements outside BRCA1 and determining whether mutations in these elements contribute to BRCA1 deregulation is still relatively unexplored. This project aims to expand the repertoire of BRCA1 long-range regulatory elements using gene conformation techniques such as chromosome conformation capture (3C) and its derivatives. The project hypothesis is that defects in long-range regulatory elements of BRCA1 transcription contribute to breast cancer predisposition.Using a candidate 3C approach, we identified a potential cis-regulatory element, called PRE1, located approximately 157 kb downstream of the BRCA1 promoter. The frequency of this interaction increased with estrogen stimulation, suggesting that chromatin looping is associated with BRCA1 transcription. Bioinformatic analysis showed that this element is flanked by DNase hypersensitivity and histone marks, which suggests it is an enhancer of BRCA1. This was confirmed by luciferase reporter assays which demonstrated that PRE1 acts as a weak enhancer, resulting in a 10% increase in BRCA1 promoter activity. Using ChIP we showed that the TF c-Myc binds to both PRE1 and the BRCA1 promoter, however depletion of c-Myc RNA levels did not alter PRE1s enhancer activity. Using a genome-wide circular-3C (4C) approach, we identified a potential trans-regulatory silencer element located 5kb upstream of G2E3 on chromosome 14. This trans-interaction was 2 validated by 3C in MCF7 cells, which suggests that the interaction is potentially functional.Consistent with this, bioinformatic analysis showed that this element, called PRE2, is flanked by DNase hypersensitivity and strong histone marks indicative of a silencer.Notably, luciferase assays showed that a modified version of PRE2 could repress BRCA1 promoter activity, resulting in a 3-fold decrease in activity. This research project has provided the first important steps to fully understanding the molecular pathways underlying BRCA1 regulation. The identification and characterisation of novel long-range control elements of breast cancer genes will immediately improve genetic screening procedures, and permit the development of more specific therapeutic approaches.3

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Partial repression of human γ-globin genes by LCR element HS3 when linked to β-globin genes and LCR element HS2 in MEL cells

Cited by 5 publications

References 35 publications

Genotypic heterogeneity and correlation to intergenic haplotype within high HbF β‐thalassemia intermedia

Genotypic heterogeneity and correlation to intergenic haplotype within high HbF β‐thalassemia intermedia

The Aγ-globin 3′ element provides no unique function(s) for human β-globin locus gene regulation

Identifying long-range control elements of the BRCA1 promoter

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