Fig 2: Model of the BRCA1-BARD1 complex-mediated TOP2B regulation. During the resting state of transcription, Pol II is paused in the promoter-proximal site in hIEGs. The pausing is induced and stabilized by various factors including transcription factors, nucleosome modifiers and nucleic acids. The BRCA1-BARD1 complex that is engaged with hIEGs interacts with and ubiquitinates TOP2B in the EGR1 TSS. This ubiquitination (marked as ub) confers an enhanced DNA-binding affinity and stability to TOP2B (Tight TOP2B). In transcriptional activation, ATM/ATR phosphorylates its substrates including BRCA1 at S1524 (phosphorylation, marked as P in a blue circle). Phosphorylated BRCA1 alters the functional interaction between BRCA1-BARD1 and TOP2B to mitigate TOP2B ubiquitination. This event destabilizes and loosens TOP2B (Loose TOP2B) from the TSS. The interplay between BRCA1-BARD1 complex and TOP2B appears to be crucial in transcriptional regulation of hIEGs. Lightning marks are proposed catalysis sites of TOP2B.

Fig 3: Gene ontology enrichment analysis reveals Top2b function in neurite growth and maintenance/survival of retinal cells.Differentially expressed genes were analyzed for gene ontology (GO) enrichment using GSEA. The result was mapped on a network of gene-sets (nodes) connected by their similarity (edges) for P0 (A) and P6 (B) using Cytoscape (p=0.005, FDR q-value =1, and similarity =0.5). Nodes highly enriched with up-regulated genes in cKO samples are shown in red while those with down-regulated genes are shown in blue. Node size represents the gene-set size. Edge thickness represents the degree of overlap between two gene-sets. Nodes were grouped according to GO definition and annotated with the group names. The cluster of red nodes under restricted cut-off values (p=0.005, FDR q-value =0.1, and similarity =0.5) was enlarged, showing that cell death, cellular transportation and neurological system development related processes were highly enriched in cKO samples.

Fig 4: Top2b deletion leads to a decreased number of photoreceptors and loss of their outer segments.(A,B) P21 retina sections of control and cKO mice were stained with DAPI. The cKO retina appeared much thinner as compare to the width of control retina. The nuclei of cells in ONL of cKO retinas were also larger than those in control retinas (enlarged). (C) Quantification of the number of ONL cells (i.e. photoreceptors) showed a dramatic decrease of these cells in cKO retinas at P14 and P21. Error bars are s.d. (n = 3). Student's t-test: *, p<0.05; **, p<0.01. (D–G) Rhodopsin staining of P14 and P21 retina sections. Rhodopsin-stained outer segment layer (OSL) was detected in the control retinas (arrows), but not in cKO retinas (arrowheads). Instead, co-staining of Rhodopsin and DAPI (inserts in E and G show higher magnification of the boxed regions) of cKO retinas indicates the expression of Rhodopsin in the cytoplasm of ONL cells. (H–K) Recoverin staining of P14 and P21 retina sections. Recoverin stained outer plexiform layer (OPL, arrows) in the controls, but not in cKO retinas (arrowheads). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OLM, outer limiting membrane; OSL, outer segment layer. Scale bars: 50 µm.

Fig 5: Aberrant retina lamination and loss of plexiform layers caused by Top2b deletion.(A) Retina sections prepared from P14, P21 and P42 control and Top2b cKO mice were stained with DAPI and co-stained with either Prox1 or Chx10. Prox1+ and Chx10+ cells were detected in the ONL (arrowheads) in Top2b cKO retinas. (B) Co-staining of E19.5-P42 control and KO/cKO retina sections with DAPI and Calretinin, which labels neurofilaments in the IPL. In cKO retinas, only a few filament-like structures were found at P7, P14 and P21, while in control samples the strata structure of neurofilaments appeared as early as P0 (arrows). (C) Co-staining with Tuj-1in P7, P14 and P21 control and cKO retina sections. Tuj-1 stained neurofilaments of the OPL (arrows) and processes of photoreceptors (arrowheads), which were largely missing in cKO samples. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 50 µm.