Detection of DNA-RNA hybrids in vivo

DNA-RNA hybrids form naturally during essential cellular functions such as transcription and replication. However, they may be an important source of genome instability, a hallmark of cancer and genetic diseases. Detection of DNA-RNA hybrids in cells is becoming crucial to understand an increasing number of molecular biology processes in genome dynamics and function and to identify new factors and mechanisms responsible for disease in biomedical research. Here, we describe two different procedures for the reliable detection of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae and in human cells: DNA-RNA Immunoprecipitation (DRIP) and Immunofluorescence

Roles of human POLD1 and POLD3 in genome stability

DNA replication is essential for cellular proliferation. If improperly controlled it can constitute a major source of genome instability, frequently associated with cancer and aging. POLD1 is the catalytic subunit and POLD3 is an accessory subunit of the replicative Pol δ polymerase, which also functions in DNA repair, as well as the translesion synthesis polymerase Pol ζ, whose catalytic subunit is REV3L. In cells depleted of POLD1 or POLD3 we found a differential but general increase in genome instability as manifested by DNA breaks, S-phase progression impairment and chromosome abnormalities. Importantly, we showed that both proteins are needed to maintain the proper amount of active replication origins and that POLD3-depletion causes anaphase bridges accumulation. In addition, POLD3-associated DNA damage showed to be dependent on RNA-DNA hybrids pointing toward an additional and specific role of this subunit in genome stability. Interestingly, a similar increase in RNA-DNA hybrids-dependent genome instability was observed in REV3L-depleted cells. Our findings demonstrate a key role of POLD1 and POLD3 in genome stability and S-phase progression revealing RNA-DNA hybrids-dependent effects for POLD3 that might be partly due to its Pol ζ interaction

Spontaneous DNA-RNA hybrids: differential impacts throughout the cell cycle

A large body of research supports that transcription plays a major role among the many sources of replicative stress contributing to genome instability. It is therefore not surprising that the DNA damage response has a role in the prevention of transcription-induced threatening events such as the formation of DNA-RNA hybrids, as we have recently found through an siRNA screening. Three major DDR pathways were defined to participate in the protection against DNA-RNA hybrids: ATM/CHK2, ATR/CHK1 and Postreplication Repair (PRR). Based on these observations, we envision different scenarios of DNA-RNA hybridization and their consequent DNA damage.European Research Council ERC2014 AdG669898 TARLOO

The DNA damage response acts as a safeguardagainst harmful DNA–RNA hybrids ofdifferent origins

Despite playing physiological roles in specific situations, DNA–RNA hybrids threat genome integrity. To investigate how cells do counteract spontaneous DNA–RNA hybrids, here we screen an siRNA library covering 240 human DNA damage response (DDR) genes and select siRNAs causing DNA–RNA hybrid accumulation and a significant increase in hybrid‐dependent DNA breakage. We identify post‐replicative repair and DNA damage checkpoint factors, including those of the ATM/CHK2 and ATR/CHK1 pathways. Thus, spontaneous DNA–RNA hybrids are likely a major source of replication stress, but they can also accumulate and menace genome integrity as a consequence of unrepaired DSBs and post‐replicative ssDNA gaps in normal cells. We show that DNA–RNA hybrid accumulation correlates with increased DNA damage and chromatin compaction marks. Our results suggest that different mechanisms can lead to DNA–RNA hybrids with distinct consequences for replication and DNA dynamics at each cell cycle stage and support the conclusion that DNA–RNA hybrids are a common source of spontaneous DNA damage that remains unsolved under a deficient DDR.European Research Council (ERC2014AdG669898TARLOOP)Worldwide Cancer Research (WCR15-00098

Multiple signaling kinases target Mrc1 to prevent genomic instability triggered by transcription-replication conflicts

Conflicts between replication and transcription machineries represent a major source of genomic instability and cells have evolved strategies to prevent such conflicts. However, little is known regarding how cells cope with sudden increases of transcription while replicating. Here, we report the existence of a general mechanism for the protection of genomic integrity upon transcriptional outbursts in S phase that is mediated by Mrc1. The N-terminal phosphorylation of Mrc1 blocked replication and prevented transcription-associated recombination (TAR) and genomic instability during stress-induced gene expression in S phase. An unbiased kinome screening identified several kinases that phosphorylate Mrc1 at the N terminus upon different environmental stresses. Mrc1 function was not restricted to environmental cues but was also required when unscheduled transcription was triggered by low fitness states such as genomic instability or slow growth. Our data indicate that Mrc1 integrates multiple signals, thereby defining a general safeguard mechanism to protect genomic integrity upon transcriptional outbursts.España, MINECO BFU2015-64437-PFEDER BFU2014-52125-REDT, and BFU2014-51672-REDC to F.P.; BFU2014-52333-P and FEDER to E.N.; and BFU2013-4291

The yeast and human FACT chromatin-reorganizing complexes resolve R-loop-mediated transcription-replication conflicts

FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP–chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription–replication conflicts, likely associated with a specific chromatin organization

Polycomb RING1A- and RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S-phase progression

The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress.We have used a cellularmodel of conditionally inactive polycomb E3 ligases (RING1A and RING1B), which monoubiquitylate lysine 119 of histone H2A (H2AK119Ub), to examine DNA replication in unperturbed cells. We identify slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with chromocenters, the sites of coalesced pericentromeric heterocromatic (PCH) domains, were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methyl- CpG binding domain protein 1. The acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1A) upregulation, could be uncoupled from a response to DNA damage. These findings link cell proliferation and the polycomb proteins RING1A and RING1B to S-phase progression through a specific function in PCH replication.Ministerio de Economı́a y Competitividad BFU2010-18146, SAF2013-47997, BFU2013-42918-

The antitumor drugs trabectedin and lurbinectedin induce transcription-dependent replication stress and genome instability

R-loops are a major source of replication stress, DNA damage, and genome instability, which are major hallmarks of cancer cells. Accordingly, growing evidence suggests that R-loops may also be related to cancer. Here we show that R-loops play an important role in the cellular response to trabectedin (ET743), an anticancer drug from marine origin and its derivative lurbinectedin (PM01183). Trabectedin and lurbinectedin induced RNA–DNA hybrid-dependent DNA damage in HeLa cells, causing replication impairment and genome instability. We also show that high levels of R-loops increase cell sensitivity to trabectedin. In addition, trabectedin led to transcription-dependent FANCD2 foci accumulation, which was suppressed by RNase H1 overexpression. In yeast, trabectedin and lurbinectedin increased the presence of Rad52 foci, a marker of DNA damage, in an R-loop–dependent manner. In addition to providing new insights into the mechanisms of action of these drugs, our study reveals that R-loops could be targeted by anticancer agents. Given the increasing evidence that R-loops occur all over the genome, the ability of lurbinectedin and trabectedin to act on them may contribute to enhance their efficacy, opening the possibility that R-loops might be a feature shared by specific cancers. Implications: The data presented in this study provide the new concept that R-loops are important cellular factors that contribute to trabectedin and lurbinectedin anticancer activity.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-PPharmaMar PRJ20140221

Depletion of the MFAP1/SPP381 Splicing Factor Causes R-Loop-Independent Genome Instability

THO/TREX is a conserved complex with a role in messenger ribonucleoprotein biogenesis that links gene expression and genome instability. Here, we show that human THO interacts with MFAP1 (microfibrillar-associated protein 1), a spliceosome-associated factor. Interestingly, MFAP1 depletion impairs cell proliferation and genome integrity, increasing γH2AX foci and DNA breaks. This phenotype is not dependent on either transcription or RNA-DNA hybrids. Mutations in the yeast orthologous gene SPP381 cause similar transcription-independent genome instability, supporting a conserved role. MFAP1 depletion has a wide effect on splicing and gene expression in human cells, determined by transcriptome analyses. MFAP1 depletion affects a number of DNA damage response (DDR) genes, which supports an indirect role of MFAP1 on genome integrity. Our work defines a functional interaction between THO and RNA processing and argues that splicing factors may contribute to genome integrity indirectly by regulating the expression of DDR genes rather than by a direct role.European ResearchCouncil (grant ERC2014 AdG669898 TARLOOP)Junta de Andalucía Spain (grant BIO1238)Spanish Ministry of Economy and Competitiveness (grant BFU2016-75058-P

Epigenetic features of human telomeres

Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, different human cell lines might carry diverse epigenetic marks at telomeres. We have developed a reliable procedure to study the chromatin structure of human telomeres independently of subtelomeres and ITSs. This procedure is based on the statistical analysis of multiple ChIP-seq experiments. We have found that human telomeres are not enriched in the heterochromatic H3K9me3 mark in most of the common laboratory cell lines, including embryonic stem cells. Instead, they are labeled with H4K20me1 and H3K27ac, which might be established by p300. These results together with previously published data argue that subtelomeric heterochromatin might control human telomere functions. Interestingly, U2OS cells that exhibit alternative lengthening of telomeres have heterochromatic levels of H3K9me3 in their telomeres.Ministerio de Economía, Industria y Competitividad de España BIO2016–78955-PConsejo Europeo de Investigación ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad de España BFU2016–75058-