Selected publications

• Brown Jill M and Buckle Veronica J (2009) FISH; Methods and Applications Humana Press. Methods in Molecular Biology.

• Brown Jill M, Green Joanne, das Neves Ricardo P, Wallace Helen AC, Smith Andrew JH, Hughes Jim, Gray Nicki, Taylor Steve, Wood William G, Higgs Douglas R, Iborra Francisco J, and Buckle Veronica J (2008) Association between active genes occurs at nuclear speckles and is modulated by chromatin environment. J Cell Biol, 182(6):1083-97.

• Hong Dengli, Gupta Rajeev, Ancliff Philip, Atzberger Ann, Brown John, Soneji Shamit, Green Joanne, Colman Sue, Piacibello Wanda, Buckle Veronica, Tsuzuki Shinobu, Greaves Mel, and Enver Tariq (2008) Initiating and cancer-propagating cells in TEL-AML1-associated childhood leukemia. Science, 319(5861):336-9.

• Iborra Francisco J and Buckle Veronica (2008) Wide confocal cytometry: a new approach to study proteomic and structural changes in the cell nucleus during the cell cycle. Histochem Cell Biol, 129(1):45-53.

• De Gobbi Marco, Viprakasit Vip, Hughes Jim R, Fisher Chris, Buckle Veronica J, Ayyub Helena, Gibbons Richard J, Vernimmen Douglas, Yoshinaga Yuko, de Jong Pieter, Cheng Jan-Fang, Rubin Edward M, Wood William G, Bowden Don, and Higgs Douglas R (2006) A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter. Science, 312(5777):1215-7.

The nucleus is a highly ordered but plastic body of chromatin and nuclear substructures, all encased by a nuclear lamina. The genome itself is organised in a non-random fashion, both three-dimensionally and with regard to linear gene distribution. Widely expressed gene-dense domains show non-random clustering, are less condensed than areas of gene paucity, and tend to occupy the interior of the nucleus. The organisation of chromatin within the nucleus has a key role to play in the processes of transcription and replication but the mechanisms governing how chromatin domains are established and altered during the course of development and differentiation remain poorly understood. Similarly, there is a broad consensus that enhancer elements exert their effect on gene promoters by direct interaction but many questions remain about what may drive such formations, how long they are stable for and how they may alter with transcriptional activity. 

Erythroblast nuclei showing positioning of alpha globin (red), beta globin (white), SLC4A1 (green) and ERAF (blue) genes. Erythroid genes associate much more frequently when active and the association occurs at nuclear speckles.

Our studies are directed at understanding the relevance of nuclear organisation to gene expression during erythropoiesis. Our ultimate aim is to gain better insight into transcriptional regulation. Erythroid cells differentiate over the course of a few days from committed blast-forming cells, through the pronormoblast stage where they are highly proliferative and begin to produce large quantities of haemoglobin, to a condensed pyknotic state when nuclei are finally extruded from the cells. During this process, erythroid-specific genes are switched on and transcribe heavily before being shut down.

Condensin subunit CAPD3 (red) in the nucleoli of a Hela cell nucleus expressing histone protein H2B-GFP (green).

We are characterising the nuclear organisation of erythroid genes and surrounding chromatin during differentiation. We find that co-transcribed erythroid genes frequently associate but see no evidence that this occurs at shared transcription foci. It seems that the aggregation of splicing factors into large nuclear speckles may be bringing active genes, particularly those on decondensed stretches of chromatin, into closer proximity.

Erythroid-specific genes, alpha globin (green) and SLC4A1 (red), sit at common SC35-enriched nuclear speckles (blue) in erythroblast nuclei.

We are particularly focussing on the organisation of chromatin around the α-globin genes on chromosome 16 short arm, looking at what changes in decondensation and interactions are necessary for transcription. To understand the spatial organisation of the α-globin gene regulatory region during commitment, differentiation and transcription on a cell-by-cell basis, we are developing systems in which we can visualise looping and transcription in real time in live cells.

Changes in nuclear activity and organisation during erythroid differentiation

The Buckle Lab Members