A Retinoblastoma Orthologue Is a Major Regulator of S-Phase, Mitotic, and Developmental Gene Expression in Dictyostelium

Abstract

BackgroundThe retinoblastoma tumour suppressor, Rb, has two major functions. First, it represses genes whose products are required for S-phase entry and progression thus stabilizing cells in G1. Second, Rb interacts with factors that induce cell-cycle exit and terminal differentiation. Dictyostelium lacks a G1 phase in its cell cycle but it has a retinoblastoma orthologue, rblA. Methodology/Principal FindingsUsing microarray analysis and mRNA-Seq transcriptional profiling, we show that RblA strongly represses genes whose products are involved in S phase and mitosis. Both S-phase and mitotic genes are upregulated at a single point in late G2 and again in mid-development, near the time when cell cycling is reactivated. RblA also activates a set of genes unique to slime moulds that function in terminal differentiation.ConclusionsLike its mammalian counterpart Dictyostelium, RblA plays a dual role, regulating cell-cycle progression and transcriptional events leading to terminal differentiation. In the absence of a G1 phase, however, RblA functions in late G2 controlling the expression of both S-phase and mitotic genes.

Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks

“…Accordingly, we predict that immediately following cell division cells will enter S-phase. Consistent with this and previous reports (Araki et al, 1994;MacWilliams et al, 2006;Strasser et al, 2012), we observe increased numbers of BrdU-stained cells immediately following cell division between 3 and 4 hours after Ax2 and adprt2 2 cells were stained with antibodies that recognise phosphorylated H2AX (cH2AX). In parallel, cells were left untreated and incubated in 100 mM BrdU for 30 minutes and subsequently subjected to immunofluorescence using antibodies against BrdU.…”

“…The cell synchronisation protocol was adapted from previously described methods (Araki et al, 1994;Strasser et al, 2012). Briefly, exponentially growing cells were seeded at between 0.75 and 1610 6 cells/ml and incubated in shaking suspension (220 rpm) at 9.5˚C for 16 hours to induce cell cycle arrest in G2.…”

“…One interpretation of these data are that in the absence of Adprt2, MMS-induced DNA damage is specifically recognised by components of the NHEJ machinery. However, given that ARTs have been implicated promoting HR at stalled and/or collapsed replication forks (Bryant et al, 2009;Sugimura et al, 2008;Yang et al, 2004), an alternative possibility is that this observation reflects a defect in the HR pathway in adprt2 2 cells, as opposed to an inherent bias towards NHEJ. However, compared to untreated control cultures, FLAG-Rad51 is enriched in chromatin fractions prepared from Ax2 and adprt2 2 cells exposed to the DSB-inducing agent phleomycin ( Fig.…”

“…It is important to note that vertebrate ARTs have been implicated in promoting HR, particularly at DSBs generated at stalled replication forks (Bryant et al, 2009;Sugimura et al, 2008;Yang et al, 2004). Given that Adprt2 is acting in a manner analogous to PARP1, at least as regards signalling base damage and DNA SSBs, an important consideration is that the absence of FLAG-Rad51 recruitment to MMS-induced DNA lesions in adprt2 2 cells reflects an inherent defect in HR, as opposed to a preferential engagement of the NHEJ machinery.…”

“…DNA DSBs can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ) (Branzei and Foiani, 2008). PARP1 has been implicated in HR-mediated repair of DSBs at stalled and/or damaged replication forks (Bryant et al, 2009;Sugimura et al, 2008;Yang et al, 2004), in addition to alternative NHEJ (A-NHEJ), an end-joining pathway that is activated in the absence of core NHEJ factors such as DNAPKCs and Ku (Audebert et al, 2004;Brown et al, 2002;Robert et al, 2009;Wang et al, 2006). However, its role in classic NHEJ is less clear.…”