MDC1 maintains active elongation complexes of RNA polymerase II.

The role of MDC1 in the DNA damage response has been extensively studied; however, its impact on other cellular processes is not well understood. Here, we describe the role of MDC1 in transcription as a regulator of RNA polymerase II (RNAPII). Depletion of MDC1 causes a genome-wide reduction in the abundance of actively engaged RNAPII elongation complexes throughout the gene body of protein-encoding genes under unperturbed conditions. Decreased engaged RNAPII subsequently alters the assembly of the spliceosome complex on chromatin, leading to changes in pre-mRNA splicing. Mechanistically, the S/TQ domain of MDC1 modulates RNAPII-mediated transcription. Upon genotoxic stress, MDC1 promotes the abundance of engaged RNAPII complexes at DNA breaks, thereby stimulating nascent transcription at the damaged sites. Of clinical relevance, cancer cells lacking MDC1 display hypersensitivity to RNAPII inhibitors. Overall, we unveil a role of MDC1 in RNAPII-mediated transcription with potential implications for cancer treatment.

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

Recent advances in the nucleolar responses to DNA double-strand breaks.

DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair.

The nucleolus is a nuclear sub-domain containing the most highly transcribed genes in the genome. Hundreds of human ribosomal RNA (rRNA) genes, located in the nucleolus, rely on constant maintenance. DNA double-strand breaks (DSBs) in rRNA genes activate the ATM kinase, repress rRNA transcription and induce nucleolar cap formation. Yet how ribosomal-DNA (rDNA) lesions are detected and processed remains elusive. Here, we use CRISPR/Cas9-mediated induction of DSBs and report a chromatin response unique to rDNA depending on ATM-phosphorylation of the nucleolar protein TCOF1 and recruitment of the MRE11-RAD50-NBS1 (MRN) complex via the NBS1-subunit. NBS1- and MRE11-depleted cells fail to suppress rRNA transcription and to translocate rDNA into nucleolar caps. Furthermore, the DNA damage response (DDR) kinase ATR operates downstream of the ATM-TCOF1-MRN interplay and is required to fully suppress rRNA transcription and complete DSB-induced nucleolar restructuring. Unexpectedly, we find that DSBs in rDNA neither activate checkpoint kinases CHK1/CHK2 nor halt cell-cycle progression, yet the nucleolar-DDR protects against genomic aberrations and cell death. Our data highlight the concept of a specialized nucleolar DNA damage response (n-DDR) with a distinct protein composition, spatial organization and checkpoint communication. The n-DDR maintains integrity of ribosomal RNA genes, with implications for cell physiology and disease.

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.