The Chromatin “Landscape” of a Murine Adult β-Globin Gene Is Unaffected by Deletion of Either the Gene Promoter or a Downstream Enhancer

Cadiz-Rivera, Brenda; Fromm, George; Vries, Christina de; Fields, Jennifer; McGrath, Kathleen E.; Fiering, Steven; Bulger, Michael

doi:10.1371/journal.pone.0092947

Cited by 6 publications

(9 citation statements)

References 27 publications

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“…2h). We find that, in concordance with detailed genetic gene competition studies at this locus [18][19][20] , partnering at the enhancer hub is allowed between developmentally synchronized genes, but not between genes active at different stages of development. The uncovered higher-order conformational features therefore provide a topological framework that helps interpreting the genetic observations.…”

supporting

confidence: 81%

“…Genetic studies in mice further demonstrated that the two developmentally distinct sets of genes compete for activation between sets, but not among members of each set, and that the four enhancer elements of the SE can compensate to a high degree for each other's activity 19,20 . We performed MC-4C experiments in both fetal liver, where the adult genes are highly expressed and in fetal brain where the b-globin locus is transcriptionally silent.…”

mentioning

confidence: 99%

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Enhancer hubs and loop collisions identified from single-allele topologies

et al. 2018

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Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the β-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect 'cohesin traffic jams'. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.

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supporting

confidence: 81%

mentioning

confidence: 99%

Enhancer hubs and loop collisions identified from single-allele topologies

et al. 2018

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“…These results strongly suggest that LCR/β min looping depends on KLF1. They also are supportive of a β maj /β min competition model, although this issue is unresolved ( 45 , 46 ).…”

Section: Resultsmentioning

confidence: 91%

LDB1-mediated enhancer looping can be established independent of mediator and cohesin

Krivega

Dean

2017

Nucleic Acids Research

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Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer–promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer–promoter looping in erythroid cells independent of mediator and cohesin.

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“…It is interesting to compare our data with other recently published examples of developmental enhancer deletion. A study of single conserved limb enhancers showed that 90% of these had no impact on cognate gene expression (21), as well as highly conserved ß-globin enhancers (22). Computational analyses have predicted that most (96%) of enhancers within the genome are tolerant to LoF mutations, and that many essential genes will have a degree of enhancer redundancy (23).…”

Section: Discussionmentioning

confidence: 99%

“…Computational analyses have predicted that most (96%) of enhancers within the genome are tolerant to LoF mutations, and that many essential genes will have a degree of enhancer redundancy (23). In the vast majority of examples, deletion of a single conserved enhancer element has no impact on cognate gene expression (21, 22). The Nestin enhancer therefore appears to be unusual in having such a profound impact on Nes expression during early embryonic development.…”

Section: Discussionmentioning

confidence: 99%

The Nestin neural enhancer is essential for normal levels of endogenous Nestin in neuroprogenitors but is not required for embryo development

Thomson

Dawson

H’ng

et al. 2021

Preprint

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Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin (Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo. We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans-chromosomal interaction of the Nes enhancer and promoter.

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The Chromatin “Landscape” of a Murine Adult β-Globin Gene Is Unaffected by Deletion of Either the Gene Promoter or a Downstream Enhancer

Cited by 6 publications

References 27 publications

Enhancer hubs and loop collisions identified from single-allele topologies

Enhancer hubs and loop collisions identified from single-allele topologies

LDB1-mediated enhancer looping can be established independent of mediator and cohesin

The Nestin neural enhancer is essential for normal levels of endogenous Nestin in neuroprogenitors but is not required for embryo development

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