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Trabajo presentado en las VIII Jornadas de Cromatina y Epigenética, de la Societat Catalana de Biologia, celebradas en Barcelona (España) el día 16 de marzo de 2018.

It is known that differentiated cells can be reprogrammed when exposed to an oocyte or an embryonic extract. In this work, we followed early reprogramming events in S2 cells treated with DREX (Drosophila early-embryo extract), and complemented this data with results obtained from S2 cells overexpressing dBigH1, together with in vitro chromatin reconstitutions using the same extract. We have observed that a simultaneous loss of somatic H1 linker histone is not necessary for the chromatin incorporation of the embrionic linker histone dBigH1. Observed decreasement in H3K4me3, H3 acetylation and changes in transcription are in correlation with previosly known information of dBigH1 being transcriptional repressor in early embryogenesis. Decrease in the number of HP1a foci in extract-incubated nuclei reflects reorganization of heterochromatin during the reprogramming process. We have also shown that dBigH1 incorporation into chromatin is ATP-facilitated. The affects of depletion dNAP-1 and dNLP histone chaperons and Acf1 subunit of the ACF chromatin remodeler have shown that dNAP-1 has a significant role in chaperoning not only dBigH1 but also H1 in this reprogramming system. The combined approach of the three systems has shown to be useful tool to follow the changes in the epigenetic landscape and in nuclear processes connected to the presence of the embrionic linker histone.

El pdf del artículo es el manuscrito de autor., PhoB is a two-component response regulator that activates transcription by interacting with the σ 70 subunit of the E. coli RNA polymerase in promoters in which the-35 σ 70-recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the σ 4 domain of σ 70 fused with the RNA polymerase β subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how σ 4 recognizes the upstream pho box repeat. As with the-35 element, σ 4 achieves this recognition through the N-terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices α2 and α3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost-35 element contacts are compensated by new ones with the activator. In addition, σ 4 is reoriented, thereby suggesting a remodelling mechanism for transcription initiation. © 2011 European Molecular Biology Organization., This study was supported by the Ministerio de Ciencia e Innovación (Grants BFU2008-02372/BMC and CONSOLIDER CSD 2006-23 to MC and BFU2009-07134 to MS), the Generalitat de Catalunya (Grants SGR2009-1309 to MC and SGR2009-1366 to MS) and the European Commission (Spine2-Complexes LSHG-CT-2006-031220 and Rapid FP7-PEOPLE-2011-ITN-290246).

13 pages, 7 figures.-- PMID: 8253082 [PubMed].-- PMCID: PMC413633., The human U1 and U6 genes have similar basal promoter structures. A first analysis of the factor requirements for the transcription of a human U1 gene by RNA polymerase II in vitro has been undertaken, and these requirements compared with those of human U6 gene transcription by RNA polymerase III in the same extracts. Fractions containing PSE-binding protein (PBP) are shown to be essential for transcription of both genes, and further evidence that PBP itself is required for U1 as well as U6 transcription is presented. On the other hand, the two genes have distinct requirements for TATA-binding protein (TBP). On the basis of chromatographic and functional properties, the TBP, or TBP complex, required for U1 transcription appears to differ from previously described complexes required for RNA polymerase I, II or III transcription. The different TBP requirements of the U1 and U6 promoters are reflected by specific association with either TFIIB or TFIIIB respectively, thus providing a basis for differential RNA polymerase selection., We wish to thank Henk Stunnenberg, Dirk Bohmann, Francis Stewart and Peter Becker for comments on the manuscript, Jacky Schmidt and H.Stunnenberg for helpful suggestions and plasmids, the EMBL photolab, Susie Weston and Sam O'Loughlin for efficient oligonucleotide synthesis, and Maryka Kimmins and Fiona Berrie for typing the manuscript. We are particularly grateful to all other members of the group for constructive criticism and suggestions. J.B. was supported by an EEC fellowship, J.D.L. by a Human Frontiers Science Program fellowship and S.I.G. by an Alexander von Humboldt fellowship.

7 pages, 4 figures.-- PMID: 2377452 [PubMed].-- PMCID: PMC331161., The homopyrimidine-homopurine sequence d(CT/GA)22 undergoes, in the presence of zinc ions, transition to an altered DNA conformation (*H-DNA) which is neither H-DNA nor B-DNA. *H-DNA is characterized by a peculiar chemical reactivity pattern in which most of the polypyrimidine strand is hyperreactive to osmium tetroxide and the central part of the polypurine strand is sensitive to diethylpyrocarbonate. Formation of *H-DNA is specific of metal-ion. *H-DNA is detected in the presence of Zn++, Cd++ and Mn++. The efficiency on promoting the transition is in the order of Zn++ greater than Cd++ much greater than Mn++. Formation of *H-DNA is also specific of nucleotide sequence. From all the different homopolymeric sequences tested only the d(CT/GA)22 sequence showed the zinc-induced transition to *H-DNA. These results suggest that stabilization of *H-DNA involves the formation of a specific complex between the metal-ion and the nucleotide sequence. The biological relevance of these results is discussed in view of the important role that zinc ions play on many nucleic acids processes., This work was financed by grants from the Comision Interministerial de Ciencia y Tecnologia (BIO-88-0236), the Comission for the European Communities (BAP-0466,E(JR) and ST2J-0372-C(A)) and the Fondo de Investigaciones Sanitarias (88/1447 and 89/510). J.B. was a recipient of a postdoctoral fellowship from the Consejo Superior de Investigaciones Cientificas. R.B. and J.M.C. were recipients of doctoral fellowships from the Ministerio de Educacion y Ciencia.

[cat] A les cèl·lules eucariotes, el DNA es troba unit a unes proteïnes bàsiques anomenades histones formant una estructura nucleoproteica que rep el nom de cromatina. Mitjançant la unió a aquestes proteïnes, el genoma queda altament empaquetat possibilitant que hi càpiga en el diminut espai del nucli cel·lular. La unitat estructural bàsica de la cromatina és el nucleosoma. Aquest està format per 146 pb de DNA que s’enrotllen al voltant d’un nucli proteic octamèric format per 2 còpies de cadascuna de les histones H2A, H2B, H3 i H4. A banda d’aquestes histones que conformen el nucli proteic del nucleosoma, també existeix una histona addicional, coneguda com a histona H1, que interacciona amb el DNA als llocs d’entrada i sortida del nucleosoma del DNA, permetent la protecció de 20 pb de bases addicionals. La histona H1 estabilitza el nucleosoma i facilita la compactació de la fibra nucleosomal en estructures de major complexitat. La biologia de la histona H1, però, resta poc estudiada. Malgrat durant molt temps ha estat considerada un repressor general de l’expressió gènica, estudis recents a Drosophila han posat de manifest que això no és del tot cert i han contribuït a revelar noves funcions importants en l’estabilitat del genoma i el desenvolupament. En aquesta tesi doctoral, es demostra que la histona H1 de Drosophila melanogaster (dH1) prevé l’acumulació d’R-loops i la inestabilitat genòmica a l’heterocromatina. En primer lloc, s’ha observat que les cèl·lules SL2 de Drosophila melanogaster deplecionades d’histona dH1 presenten un increment de la fosforilació de la variant H2Av a la serina 137 (γH2Av), senyal epigenètica associada a la presència de dany al DNA. En relació amb això, també s’ha vist que aquestes cèl·lules presenten una elevada inicidència de trencaments al DNA i intercanvis entre cromàtides germanes. En segon lloc, s’ha vist que aquests defectes tenen lloc preferentment a l’heterocromatina i són deguts a l’acumulació d’R-loops, unes estructures de triple cadena d’àcids nucleics compostes per un híbrid RNA:DNA i una cadena desplaçada de DNA de cadena senzilla que representen una font important d’inestabilitat genòmica. En tercer lloc, mitjançant l’anàlisi del contingut en R-loops i fosforilació d’H2Av a la serina 137 (γH2Av) al llarg del cicle cel·lular, també s’ha observat que en deplecionar la histona dH1 es formen R-loops durant la fase G1 del cicle cel·lular i que la generació de dany al DNA té lloc preferentment durant la fase S, és a dir, durant la replicació del DNA. En relació amb això últim, s’ha vist que la replicació del DNA està afectada i que aquesta afectació és deguda a la presència d’R-loops. Concretament, la replicació del DNA és més lenta i presenta una tendència més alta a aturar-se en aquestes condicions. Finalment, els resultats obtinguts també demostren que la prevenció de l’acumulació dels R-loops a l’heterocromatina podria tractar-se d’una funció específica de la histona dH1, donat que la depleció d’HP1a, un component estructural de l’heterocromatina, no té el mateix efecte. En deplecionar HP1a, no s’observa ni una acumulació d’R-loops ni de dany al DNA. Per tot això, els nostres resultats inidiquen que la histona dH1 prevé l’estrés replicatiu induit pels R-loops a l’heterocromatina i demostren que la seva contribució és essencial per al manteniment de l’estabilitat del genoma, una funció que està altament compromesa en el càncer i és de vital importancia per a la cèl·lula i la viabilitat de l’organisme., [eng] Linker histone H1 is an intrinsic component of chromatin that plays an important structural role by stabilizing the nucleosome and compacting the nucleofilament into higher-order structures. The biology of histone H1 remains, however, poorly understood. Here we show that Drosophila histone H1 (dH1) prevents genome instability as indicated by the increase of γH2Av (H2Av phosphorylated at Ser137) and the high incidence of DNA breaks (DBs) and sister-chromatid exchanges (SCE) observed in dH1depleted cells. These defects occur at heterochromatin and are due to the accumulation of R-loops, DNA:RNA hybrids that are a source of genome instability. We also show that, concomitantly to upregulation of transposons and other heterochromatic DNAs, R-loops form at G1-phase and induce γH2Av during DNA replication. dH1-depleted cells also show abnormally early heterochromatin replication and increased replication fork stalling. Furthermore, our results also suggest that prevention of R-loop-induced genome instability in heterochromatin could be histone dH1 specific, since depletion of HP1a, an intrinsic component of heterochromatin, does not result in the same phenotype. Altogether, our results indicate that linker histone H1 prevents R-loops-induced replication stress in heterochromatin and unveil its essential contribution to maintenance of genome stability, a function that is highly compromised in cancer and is central for cell and organismal viability.