Regulation of DNA Topoisomerase IIβ through RNA-dependent Association with Heterogeneous Nuclear Ribonucleoprotein U (hnRNP U)

Abstract

Recent studies suggest that DNA topoisomerase II␤ (topo II␤) is involved in transcriptional activation of certain genes, which assumes accurate targeting of the enzyme to its action site. The target selection may be achieved by cooperation with unknown regulatory factors. To seek out such factors, we looked for proteins associated with the enzyme in differentiating cerebellar neurons. Antibody against topo II␤ co-precipitated RNA-binding proteins including PSF, NonO/p54nrb, as well as hnRNP U/SAF-A/SP120. Reconstitution experiments with tag-purified proteins showed that topo II␤ associates stoichiometrically with SP120 in the presence of RNA that was co-purified with SP120. The most effective RNA species for the complex formation was a subset of cellular polyadenylated RNAs. The C-terminal 187-residue domain of SP120 was necessary and sufficient for the association with both topo II␤ and the endogenous RNA. The RNA isolated from the tag-purified SP120 inhibited the relaxation of supercoiled DNA by topo II␤. When the enzyme associates with SP120, however, the inhibition was abolished and the catalytic property was modulated to more processive mode, which may prolong its residence time at the genomic target site. Furthermore, the presence of SP120 was required for the stable expression of topo II␤ in vivo. Thus, SP120 regulates the enzyme in dual ways. DNA topoisomerase II (topo II)2 can be described as an enzyme with two different catalytic activities, deoxyribonuclease and DNA ligase. Between the cleavage and religation of one duplex DNA (G-segment), another duplex (T-segment) is transferred through the gate between the cleaved ends that are held through a covalent linkage to the tyrosine residues of two identical subunits (1). Multiple cycles of these events dissipate the supercoiling or intertwining structures in substrate DNA. Thus, the enzyme plays a major role in cellular processes such as DNA replication, transcription, and recombination (2). Topo II orthologs are widely distributed in organisms from bacteria to mammals and two paralogs, named ␣ and ␤, were first identified in human cells (3). These paralogs (isozymes) that are present in most vertebrates show similar enzymatic properties in vitro but their expression is regulated quite differently. Topo II␣ is essential in cell proliferation because it catalyzes the segregation of daughter chromosomes at the mitotic phase. This role is played by a unique topo II in lower eukaryotes. In contrast, the biological function of topo II␤ had been totally ambiguous until we compared the isozyme expression patterns in developing cerebellar cortex (4, 5). As expected, topo II␣ is the predominant isozyme expressed in proliferating precursors of granule neurons. When cells begin to differentiate after the final cell division, the isozyme expression pattern switches from topo II␣ to topo II␤. Later studies with cultured granule neurons and topo II-specific inhibitors revealed that the activity of topo II␤ is required for the transcriptional activation of a subset of g...

Genomic Regions Targeted by DNA Topoisomerase IIβ Frequently Interact With a Nuclear Scaffold/Matrix Protein hnRNP U/SAF‐A/SP120

“…It would be reasonable to assume that the nucleoplasmic topo IIβ also stays in a repressed state unless the inhibitory effect of RNA is removed by some factor(s). At present, SP120 is the likeliest candidate for such factors since the protein abolishes the inhibition by RNA by forming an RNA‐dependent stoichiometric complex with topo IIβ [Kawano et al, ]. Although it remains to be demonstrated, we speculate that topo IIβ/SP120 molecular complex interacts preferentially with AT‐rich gene‐poor genomic regions, which is consistent with the topo IIβ target clones obtained in the present study and the frequent occurrence of SP120‐binding sites in the target clones.…”

“…The topo IIβ target regions may also be enriched in binding sites for hnRNP U/SAF‐A/SP120. This protein is shown to be abundant in the nuclear scaffold/matrix (NS/M) fraction, selectively binds with S/MAR [Romig et al, ; Tsutsui et al, ], and forms an RNA‐dependent molecular complex with topo IIβ [Kawano et al, ].…”

SAF-A Forms a Complex with BRG1 and Both Components Are Required for RNA Polymerase II Mediated Transcription

“…Moreover, both SAF-A and BRG1 have been reported to interact with actin [16], [37] and DNA topoisomerase IIβ [38], [39]. In combination with these reports, and the observation that transcription is preferentially regulated at the nuclear periphery in embryonic stem cells [40], our discoveries allow us to speculate about possible roles for a SAF-A/BRG1 complex in gene activation.…”

Nuclear to Cytoplasmic Translocation of Heterogeneous Nuclear Ribonucleoprotein U Enhances TLR-Induced Proinflammatory Cytokine Production by Stabilizing mRNAs in Macrophages

“…Mice carrying a hypomorphic mutation in the hnRNP U gene exhibit postimplantation lethality (9), suggesting that hnRNP U contributes to a variety of essential biological functions, including transcriptional regulation and RNA metabolism. hnRNP U has been shown to interact with various transcriptional cofactors and modulate their transcriptional activation, such as transcriptional coactivator p300 (10), glucocorticoid receptor (11), Yes-associated protein (12), heterochromatin protein 1a (13), DNA topoisomerase II b (14), and PCAF (15). hnRNP U also can interact with the SCF b -TrCP ubiquitin ligase complex to regulate its E3 ligase activity (16,17).…”