Biology subjects

Sloane-Stanley, J. A.

Publications and source records attributed to Sloane-Stanley, J. A..

3 recordsLinked to original sources

bioRxiv2021

On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse -globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of -globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as -globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

molecular biology↗

bioRxiv2020

A functional overlap between actively transcribed genes and chromatin boundary elements

Mammalian genomes are subdivided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an individual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a [~]70 kb sub-TAD containing the duplicated mouse -like globin genes and their five enhancers (5-R1-R2-R3-Rm-R4-1-2-3). The 5 border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the -globin locus leaves the sub-TAD largely intact. The predominant 3 border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed 2-globin gene rather than the CTCF sites at the 3-end of the sub-TAD. Of interest, the almost identical 1- and 2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal 1-globin gene which behaves as a partial boundary between the enhancers and the distal 2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements. Significance StatementMammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers/promoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.

genomics↗

bioRxiv2020

Identification of MAZ as a novel transcription factor regulating erythropoiesis

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors. Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a transcription factor that binds to the promoter of the erythroid-specific human -globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ in primary human erythroid cells impairs erythroid differentiation, and genetic variants in the MAZ locus are associated with clinically important human erythroid traits. Taken together, these findings reveal the Zinc-finger transcription factor MAZ to be a previously unrecognised regulator of the erythroid differentiation program.

molecular biology↗