Treacle Sticks the Nucleolar Responses to DNA Damage Together

Abstract

The importance of chromatin environment for DNA repair has gained increasing recognition in recent years. The nucleolus is the largest sub-compartment within the nucleus: it has distinct biophysical properties, selective protein retention, and houses the specialized ribosomal RNA genes (collectively referred to as rDNA) with a unique chromatin composition. These genes have high transcriptional activity and a repetitive nature, making them susceptible to DNA damage and resulting in the highest frequency of rearrangements across the genome. A distinct DNA damage response (DDR) secures the fidelity of this genomic region, the so-called nucleolar DDR (n-DDR). The composition of the n-DDR reflects the characteristics of nucleolar chromatin with the nucleolar protein Treacle (also referred to as TCOF1) as a central coordinator retaining several well-characterized DDR proteins in the nucleolus. In this review, we bring together data on the structure of Treacle, its known functions in ribosome biogenesis, and its involvement in multiple branches of the n-DDR to discuss their interconnection. Furthermore, we discuss how the functions of Treacle in ribosome biogenesis and in the n-DDR may contribute to Treacher Collins Syndrome, a disease caused by mutations in Treacle. Finally, we outline outstanding questions that need to be addressed for a more comprehensive understanding of Treacle, the n-DDR, and the coordination of ribosome biogenesis and DNA repair.

Keywords: DNA damage response; DNA repair; Treacher Collins syndrome; chromatin; n-DDR; nucleolus; rDNA; treacle/TCOF1.

FIGURE 1

Treacle structure and cellular localization. (A) The structure of the most common isoform of Treacle. Treacle isoform d (1488 amino acids, 152 kDa, NP_001128715.1) is encoded by the Treacle transcript variant 4, containing 27 exons. It is an intrinsically disordered protein with N- (exons 1–6, aa 1–213) and C-terminal (exons 17–26, aa 954–1,488) regions, and 11 central repeat domains (exons 6A–16, aa 214–953). The N-terminal harbors a LisH domain (aa 6–38), and contains a nuclear localization signal (NLS, aa 74–77). The SDT-like motifs SETE (aa 171–174), SEDT (200–203), SDET (207–210), are responsible for NBS1 binding, and PLK interaction site was also mapped to the N-terminal domain. The 11 central repeats contain several CK2 and ATM/ATR phosphorylation sites. The central region is responsible for Pol II interaction, and aa 526–953 mediates Pol I binding. Repeat domain 6A furthermore contains a NLS but 6A is not present in all isoforms. The C-terminal region is important for Treacle localization, it contains several potential NLSs (aa 1,362–1,482). The last 41 aa of the protein mediates nucleolar localization. The C-terminal furthermore mediates rDNA and UBF binding (aa 1,294–1,488). It contains several SQ/TQ phospho motifs, three serines (S1227, S1228, S1236) responsible for TOPBP1 recruitment, and a TRF2 interaction site. Created with BioRender.com. (B) Immunofluorescence images of Treacle and UBF in the dense fibrillar component, a sub-compartment of the nucleolus where newly transcribed rRNA is located. The nucleolar area is outlined based on lower intensity of the DNA (DAPI) staining.

FIGURE 2

Cellular functions of Treacle. (A) Treacle nucleolar functions: i) Normal transcription. Treacle promotes rRNA transcription by recruiting Pol I and UBF to the rDNA promoter. By interacting with NOP56, it also mediates rRNA methylation, essential for mature rRNAs and ribosome production. ii) DSB repair. Upon DSB induction ATM becomes activated and phosphorylates Treacle, leading to Pol I transcriptional inhibition. Treacle in turn recruits the MRN complex and TOPBP1, which then activates ATR. ATR activation is required for complete rDNA transcriptional shut-down, it promotes break clustering, and their eventual translocation to nucleolar caps. In the caps, breaks become accessible to HR factors and repaired. iii) Osmotic stress. Hypoosmotic stress leads to the stabilization of nucleolar R-loops, recruiting RPA and TOPBP1. Treacle is indispensable for TOPBP1 recruitment, and subsequent ATR activation. ATR is the main kinase controlling the response, but ATM activation and NBS1 were also required for Pol I inhibition. iv) Replication stress (RS). RS leads to fork stalling in the rDNA, and activates ATR, which in turn mediates TOPBP1 recruitment through Treacle. TOPBP1 and Treacle form large foci, and promote the recruitment of RS factors, such as RPA, BRCA1 and FANCD2 in the nucleoli. By these means, stalled forks are protected, and RS-mediated rDNA breaks are minimized. v) Oxidative stress. Neuroepithelial cells have high levels of ROS, harmful to the DNA. The rDNA is normally protected by Treacle, however, upon Treacle deficiency, high levels of ROS lead to DSB formation, and consequent rRNA transcriptional inhibition. Nucleolar stress leads to the stabilization of p53 and apoptosis, causing craniofacial defects in TCS patients. When Treacle deficient cells are treated with the ROS scavenger NAC, less DNA damage and apoptosis are observed and craniofacial features are preserved in mice. (B) Treacle telomeric functions. i) Treacle is recruited to the telomeric repeats by TRF2, and it controls TERRA transcription in S-phase. Treacle interacts with Pol II and regulates its transcription. ii) TERRA levels increase upon Treacle depletion, leading to R-loop formation and telomere problems. When Treacle is absent, more Pol II associates with the telomeres, and accelerates TERRA transcription. (C) Treacle mitotic function. i) Treacle is phosphorylated by PLK1 and localizes to the kinetochores and centrosomes, promoting normal mitosis. ii) In the absence of Treacle, cells have problems with mitotic spindle assembly, chromosome alignment and mislocalization of PLK1. Created with BioRender.com.

FIGURE 3

Treacle in Treacher Collins syndrome. (A) Treacle proficient mice show normal rRNA transcription, proliferation and apoptosis in neural crest cells (NCC), and develop normal craniofacial features. (B) In mice with Treacle haploinsufficiency (tcof1 ^+/–), however, increased apoptosis was observed in NCC. Mutations in Treacle compromise rRNA transcription, leading to ribosomal protein-mediated sequestration of MDM2, and consequent p53 stabilization. Increased rDNA damage in the absence of Treacle-mediated repair can also facilitate p53 stabilization. Activation of p53 leads to increased apoptosis of NCC and to the development of craniofacial malformations. Created with BioRender.com.