The Tousled-like kinases regulate genome and epigenome stability: implications in development and disease

The Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine–threonine kinases that have been implicated in DNA replication, DNA repair, transcription, chromatin structure, viral latency, cell cycle checkpoint control and chromosomal stability in various organisms. The functions of the TLKs appear to depend largely on their ability to regulate the H3/H4 histone chaperone ASF1, although numerous TLK substrates have been proposed. Over the last few years, a clearer picture of TLK function has emerged through the identification of new partners, the definition of specific roles in development and the elucidation of their structural and biochemical properties. In addition, the TLKs have been clearly linked to human disease; both TLK1 and TLK2 are frequently amplified in human cancers and TLK2 mutations have been identified in patients with neurodevelopmental disorders characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. A better understanding of the substrates, regulation and diverse roles of the TLKs is needed to understand their functions in neurodevelopment and determine if they are viable targets for cancer therapy. In this review, we will summarize current knowledge of TLK biology and its potential implications in developmen

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.Work in I.A.’s group is funded by the WellcomeTrust (grant number 210634), BBSRC (BB/R007195/1), and Cancer ResearchUK (C35050/A22284). Work in D.A.’s group is funded by the Cancer ResearchUK Career Development Fellowship (grant number 16304). Work in the S.J.B.lab is supported by the Coun, which receives its core fundingfrom Cancer Research UK (FC0010048), the UK Medical Research Council(FC0010048), and the Wellcome Trust (FC0010048); a European Research Council (ERC) Advanced Investigator Grant (TelMetab); and Wellcome TrustSenior Investigator and Collaborative Grants. S.S.-B. was the recipient of an EMBO Long Term Fellowship (ALTF 707-2019) and a MSCA individual fellow-ship (grant 886577). Work in the J.R.C. group is funded by CRUK Career Devel-opment Fellowship (C52690/A19270) with infrastructural support from Well-come core award 090532/Z/09/ZS

Differential requirements for Tousled-like kinases 1 and 2 in mammalian development

The regulation of chromatin structure is critical for a wide range of essential cellular processes. The Tousled-like kinases, TLK1 and TLK2, regulate ASF1, a histone H3/H4 chaperone, and likely other substrates, and their activity has been implicated in transcription, DNA replication, DNA repair, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function during mammalian development and suggests that TLK1 and TLK2 have largely redundant roles in genome maintenance

Role of the Tousled like kinases in maintaining genome and epigenome stability

[eng] Histone deposition during DNA replication, transcription and repair ensures the faithful maintenance of genetic and epigenetic information. The histone H3-H4 chaperone ASF1 participates in both replication dependent and independent nucleosome assembly. While much attention has been focused on the role of histone modifications, the significance of histone chaperones and their regulation in chromatin maintenance remains relatively unexplored. The Tousled like kinases 1 and 2 (TLK1 and TLK2) are evolutionarily conserved Ser/Thr kinases that regulate ASF1. TLKs have been implicated in DNA repair and chromosome stability but their cellular functions remain poorly defined. Apart from ASF1, the DNA damage signaling protein RAD9 is another proposed target of TLK activity but the extent to which they both rely on TLK1/2 has not been clearly established. The goal of this thesis is to characterize the relative functions of TLK1 and TLK2, identify their cellular targets, modes of regulation and consequences of their deficiency during development and in cancer. To this end, we have generated conditional mouse models with loss of function alleles, studied mouse-derived cells from homeostatic and cancerous tissues, and performed siRNA mediated depletion of TLK1/2 in multiple human cell lines. In vivo, TLK1 and TLK2 play largely redundant roles in genome maintenance and cell viability in tissue homeostasis, with the exception of a placental specific requirement for TLK2. Depletion of TLK activity in cells leads to reduced histone deposition, DNA replication stress, extensive DNA damage accumulation and arrest or cell death. In addition, TLK deficient cells exhibit synthetic lethality with several DNA damage checkpoint inhibitors, consistent with a role for TLK activity in replication stress tolerance. Genome wide analysis indicated that TLK activity is required for heterochromatin maintenance, particularly in regions of repetitive DNA. This is consistent with defects in histone variant deposition and increased non-coding RNA transcription at these sites. While the phenotypes of TLK depletion support a major role in ASF1-mediated histone deposition, our data indicates that they likely control additional targets and suggests that the identification of small molecule inhibitors for these kinases could suppose a valuable target for cancer therapy to augment existing strategies for cancer treatment.[cat] La deposició d’histones durant els processos de replicació, transcripció i reparació de l’ADN asseguren el manteniment de la informació genètica i epigenètica. ASF1 és una xaperona d’histones H3-H4 que participa en la deposició d’histones tant en processos de replicació com en processos independents de replicació. Mentre que les modificacions epigenètiques de les histones han estat molt estudiades, la importància de les xaperones d’histones i la seva regulació són àmbits menys explorats. Les Tousled like kinases 1 i 2 (TLK1 i TLK2) són quinases conservades evolutivament que regulen ASF1. Les TLKs tenen un paper en el manteniment de l’estabilitat del genoma malgrat les seves funcions cel·lulars no estiguin ben definides. A banda d’ASF1, un altre substrat de les TLKs és la proteïna RAD9, implicada en senyalització en resposta a dany de l’ADN. L’objectiu d’aquesta tesi és caracteritzar les funcions relatives de TLK1 i TLK2, identificar els seus substrats, entendre com es regulen i les conseqüències de la seva deficiència durant desenvolupament i en processos tumorals. Per assolir aquests objectius, hem generat models de ratolí amb al·lels de genoanul∙lació condicional, hem estudiat la deficiència de les TLKs en cèl·lules derivades de ratolí i en línies cel·lulars humanes. In vivo, TLK1 i TLK2 tenen papers redundants en el manteniment dels teixits en homeòstasi, amb l’excepció de la placenta on hi ha un requeriment específic per la funció de TLK2. Depleció de l’activitat TLK en cèl·lules causa una reducció en deposició d’histones, estrès replicatiu, acumulació de dany a l’ADN i aturada del cicle cel·lular o mort cel·lular. La deficiència d’activitat TLK causa letalitat quan es combina amb inhibidors de CHK1 o de PARP, consistent amb un paper de les TLKs en la tolerància de l’estrès replicatiu. Un anàlisi a nivell genòmic ens demostra que l’activitat TLK és necessària per mantenir les regions d’heterocromatina, en particular regions repetitives. Aquest fenotip va acompanyat de defectes en la deposició d’una variant d’histona H3 i un increment de transcripció de RNA no codificant. Els nostres resultats recolzen un paper principal de les TLKs mitjançant el substrat ASF1, tanmateix considerem que les TLKs molt probablement controlin altres proteïnes. Finalment, suggerim que la identificació de molècules inhibidores de l’activitat TLK podrien suposar un avenç terapèutic en càncer per a augmentar l’efecte de teràpies existents

Análisis cualitativo de la viabilidad de los programas de prevención de la enfermedad y promoción de la salud en contextos de pobreza

“Análisis Cualitativo de la viabilidad de los programas de prevención de la enfermedad y promoción de la salud, en contextos de pobreza”, es una investigación realizada en la Unidad Primaria D Atención Emaus, ubicada en la localidad de Engativá, distrito capital Bogotá, que emplea los métodos cualitativos y cuantitativos de forma complementaria, para demostrar que tres programas de prevención de la enfermedad y promoción de la salud, (nutrición, salud oral y salud sexual y reproductiva), implican un gasto elevado para los hogares que asisten a las capacitaciones brindadas por estos programas. En consecuencia, tales programas no conseguirían el objetivo para el que fueron formulados, de acuerdo con las conclusiones emanadas de este estudio. Basándose en los resultados obtenidos, se empleó el materialismo dialéctico para dirigir una crítica a las nociones neoclásicas que sustentan tales programas que son aceptadas en primera instancia en este trabajo para posteriormente señalar sus contradicciones y limitaciones. / Abstract. “Qualitative analysis of the viability of the programs of prevention of the disease and promotion of the health, in poverty contexts”, is an investigation realised in Primary Unit D Emaus Attention, located in the locality of Engativá, capital district Bogota, that uses the qualitative and quantitative methods of complementary form, to demonstrate that three programs of prevention of the disease and promotion of the health, (nutrition, oral health and sexual and reproductive health), imply a cost lifted for the homes that attend the qualifications offered by these programs. Consequently, such programs would not secure the objective for which they were formulated, in agreement with the emanated conclusions of this study. Being based on the obtained results, the dialectic materialism was used to direct a critic to the neoclassic slight knowledge that sustain such programs that are accepted in first instance in this work later to indicate to their contradictions and limitations.Maestrí

RTEL1 Regulates G4/R-Loops to Avert Replication-Transcription Collisions

Regulator of telomere length 1 (RTEL1) is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1-deficient cells remains unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1 -/- cells possess G-quadruplex (G4)-DNA-forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine). Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1 -/- cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1 -/- and Rtel1 PIP_box knockin cells and in wild-type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication

Molecular basis of Tousled-Like Kinase 2 activation

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs.Novo Nordisk Foundation Center for Protein Research is supported fi nancially by the Novo Nordisk Foundation (Grant NNF14CC0001) and the Danish Cancer Foundation for a grant to G.M. T.H.S. is supported by the Ministerio de Economía y Competitividad (MINECO) (BFU2015-68354, Ayudas para incentivar la incorporación estable de doc- tores (IED) 2015 and institutional funding through the Centres of Excellence Severo Ochoa award and from the CERCA Programme of the Catalan Government), S.S.B. by a predoctoral fellowship from Fundacio La Caixa and M.V.P. by a Severo Ochoa FPI predoctoral fellowship (MINECO). P.R. is supported by the Marie Sk ł odowska-Curie European Training Network (ETN) “ TEMPERA. We would like to thank the Protein Production Facility Platform at CPR for the excellent technical assistance, M. Orozco at IRB Barcelona for helping with the initial modelling analysis and also thank the PRO-MS Danish National Mass Spectrometry Platform for Functional Proteomics and the CPR Mass Spectrometry Platform for instrument support and assistance

SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance

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