1L TERRA acts in cis and leads to shortening of telomere 1L.

<p>(A) 1L TERRA expression leads to shortening of telomere 1L. <i>Upper panel:</i> DNA was extracted from five independent clones of the TetO7-1L strain grown at 30°C on YPD plates with (+) or without (−) Dox for 25 generations and analyzed by telomere PCR for telomere 1L on a 2.5% agarose gel. Marker (M) is given in bp. <i>Lower panel:</i> 1L telomere PCR products were TOPO cloned and sequenced. Shown is the average length of the TG-tract under inducing (−Dox) and non-inducing (+Dox) conditions with standard deviations. 10 sequences were analyzed for each condition. (B) Telomere 6R is not affected by expression of 1L TERRA. Telomere PCR for telomere 6R performed with DNA from (A). (C) 1L TERRA expression does not affect the average length of Y′ containing telomeres. DNA was extracted from the indicated strains grown as in (A) and digested with <i>Xho</i>I before Southern blot analysis. The Southern blot was hybridized with a 5′ end-radiolabeled CA oligonucleotide detecting telomeric TG repeats of yeast telomeres. The telomeric TG repeat signals are highlighted with a line (<i>Xho</i>I fragment). Marker (M) is given in bp.</p

Development of an inducible 1L TERRA expression system.

<p>(A) Scheme of telomere 1L in wild type, the TetO7-1L and the control strains. +1 in the wt marks the transcription start site of 1L TERRA mapped in <i>sir3</i>Δ (see B, C). Numbers give the distance of the +1 site, the X-core element, or the <i>URA3</i> marker from the TG repeat sequence (TG_(1–3)). The arrows towards the right mark the position of the subtelomeric oligonucleotide (oBL1180, see C) used for telomere PCR of telomere 1L. Strain TetO7-1L was constructed by introducing a sequence containing <i>URA3</i>, the <i>ADH1</i>-terminator (Ter), seven TetO boxes (TetO7) and a cytochrome 1 (<i>CYC1</i>) sequence upstream of the 1L TERRA transcription start site. +1 marks the transcription start site of the inducible 1L TERRA in strain TetO7-1L (see C, D). Presence of Dox inhibits 1L TERRA expression in TetO7-1L. The control strain was constructed by insertion of the <i>URA3</i> cassette and the <i>ADH1</i>-terminator upstream of the +1 start site, without the TetO7 and <i>CYC1</i> promoter sequences. (B) Nested PCR of 5′RACE (absence (−) or presence (+) of Tobacco Acid Pyrophosphatase (TAP): removes the 5′ cap structure of the RNA) performed in <i>sir3</i>Δ for mapping the transcription start site (+1) of 1L TERRA. Marker (M) is given in base pairs. (C) Sequence of telomere 1L in strain TetO7-1L. The <i>CYC1</i> sequence upstream of the +1 start site of 1L TERRA in wt (bold and underlined) is shown in italics. The putative TATA boxes of the <i>CYC1</i> sequence are underlined <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002747#pgen.1002747-Guarente1" target="_blank">[44]</a>. The +1 start site of 1L TERRA in TetO7-1L is shown in bold, and underlined. The X-core sequence is highlighted in grey. Marked are the oligonucleotides used for the RT of the 5′RACE (oBL1181; antisense strand), for telomere PCR of telomere 1L (oBL1180; sense strand), for the nested PCR (oNI55; antisense strand), and for 1L TERRA detection by qRT-PCR (oNI54 (sense strand), oBL1181) in the X-repeat sequence (downstream of the X-core sequence). (D) Nested PCR of 5′RACE (as in B) performed on two independently generated clones of strain TetO7-1L for mapping the transcriptional start site (+1) of 1L TERRA.</p

Absence of telomerase increases the shortening of telomere 1L upon 1L TERRA expression.

<p>(A) DNA was extracted from TetO7-1L, and <i>est1</i>Δ/TetO7-1L (four independent clones) strains grown for 25 generations (gen) at 25°C on YPD plates containing 10 µg/ml Dox (lanes 3, 6, 9, 12), before plating them on YPD plates with (+) (lanes 5, 8, 11, 14) or without (−) Dox (lanes 4, 7, 10, 13) for additional 25 generations. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (B) 1L TERRA expression does not accelerate shortening of telomere 6R in the absence of telomerase. Telomere PCR for telomere 6R performed with DNA from the same strains and growth conditions as in (A). (C) Recruitment of Est2 is not affected by expression of 1L TERRA. Yeast strains were grown for 25 generations at 30°C on YPD plates with (+) or without (−) Dox. Est2-myc associated chromatin was immunoprecipitated after an additional growth to exponential phase in rich medium (−/+Dox) at 30°C (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002747#s4" target="_blank">Materials and Methods</a>). The immunoprecipitated telomeres 1L, 7L and 15L were quantified by real-time PCR and expressed as percentage of input. A mock IP lacking the myc-antibody served as negative control. Values of two independent biological replicates with standard deviation are shown.</p

1L TERRA expression depends on the Dox concentration and does not affect other TERRA species.

<p>(A) 1L TERRA expression can be modulated by the amount of Dox. qRT-PCR analysis of 1L TERRA in wt, TetO7-1L (two independent clones: cl1 and cl2), and control strains (two independent clones: cl1 and cl2) grown with different Dox concentrations to exponential phase at 30°C in rich medium. −ΔΔCT values of strains grown in 1, 0.1, 0 µg/ml Dox, normalized against actin with standard deviations are shown. The −ΔΔCT values corresponding to each strain grown in 10 µg/ml Dox is arbitrarily set to 0. (for calculations see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002747#s4" target="_blank">Materials and Methods</a> published online). (B) Expressed 1L TERRA has a size of 500–800 nt. RNA was extracted from the indicated strains (as in A) grown to exponential phase at 30°C in rich medium. 15 µg of RNA was loaded per lane on a 1.2% formaldehyde/agarose (FA) gel and analyzed by Northern blot analysis. The Northern blot was hybridized with a 5′ end-radiolabeled CA oligonucleotide detecting telomeric GU repeats in 1L TERRA. The 1L TERRA signal is marked with a line. An asterisk marks an unspecific band detected with this probe. RNA marker sizes are indicated at the left in nt. (C) The size of 1L TERRA expressed from TetO7-1L is comparable to the size of TERRA species expressed from other X-only telomeres in a <i>sir3</i>Δ mutant. RNA was extracted from the indicated strains grown to exponential phase at 30°C in rich medium. The temperature-sensitive <i>rat1-1</i> mutant, wt and <i>sir3</i>Δ strains were grown at 25°C to exponential phase in rich medium before the culture was split and either shifted to 37°C or maintained at 25°C for 1 h followed by RNA extraction. 15 µg of RNA was analyzed by Northern blot as described in (B). TERRA transcribed from Y′ and X-only telomeres (all TERRA) are enriched in <i>rat1-1</i> cells at non-permissive temperature. <i>sir3</i>Δ specifically increases TERRA levels transcribed from X-only telomeres, such as telomere 1L, independently of the temperature <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002747#pgen.1002747-Iglesias1" target="_blank">[33]</a>. The asterisk marks an unspecific band. RNA marker sizes are given to the left in nt. (D) 1L TERRA expression does not affect the levels of TERRA species transcribed from other telomeres. qRT-PCR analysis of TERRA transcribed from X-only telomeres 1L, 7L, 15L, or from 6 different Y′ telomeres (6*Y′). The indicated strains were grown in rich medium with (+) or without (−) Dox to exponential phase at 30°C. Average −ΔΔCT values of three independent biological replicates normalized against actin with standard deviation are shown. −ΔΔCT values of each strain grown in +Dox is arbitrarily set to 0.</p

Exo1 mediates shortening of telomere 1L upon 1L TERRA expression, while Mre11 is not involved.

<p>(A) TERRA induced shortening of telomere 1L is abolished by deletion of exonuclease 1 (<i>exo1Δ</i>). DNA was extracted from four independent clones of the indicated strains grown at 30°C for 25 generations on YPD plates with (+) or without (−) Dox. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (B) Length of telomere 6R is not affected by 1L TERRA expression in the absence of Exo1. Telomere PCR for telomere 6R performed with DNA from (A). (C) Deletion of Exo1 rescues increased telomere shortening of telomere 1L upon TERRA induction in the absence of telomerase. <i>est1</i>Δ/TetO7-1L (four independent clones) and <i>est1</i>Δ/<i>exo1</i>Δ/TetO7-1L (three independent clones) strains were grown for 25 generations at 25°C on YPD plates containing 10 µg/ml Dox, before plating them on YPD plates with (+) (lanes 2, 4, 6, 8, 10, 12, 14) or without (−) (lanes 1, 3, 5, 7, 9, 11, 13) Dox for additional 25 generations. Telomere lengths at 1L were analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp. (D) Telomere shortening by 1L TERRA expression is not mediated by Mre11. DNA was extracted from four independent clones of the indicated strains grown at 30°C for 25 generations on YPD plates with (+) or without (−) Dox. Telomere length at 1L was analyzed by telomere PCR on a 2.5% agarose gel. Marker (M) is given in bp.</p

Impaired neuronal maturation of hippocampal neural progenitor cells in mice lacking CRAF

<div><p>RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons.</p></div

Cell cycle abnormalities in BrdU-labelled NPCs of postnatal CRAF ko mice.

<p>(A) Immuno-histological analysis of BrdU (green) and Ki67 (red) stained sagittal brain sections of CRAF ct and CRAF ko hippocampus at P23 2h after a single BrdU application. Representative brain sections of CRAF ct (upper panel) and CRAF ko (lower panel) show BrdU-labelled cells (green) colocalizing with Ki67 (red) (merge, white arrows). White arrowheads indicate BrdU-labelled cells (green) of CRAF ko that lack any positive Ki67 (red) staining (lower panel). Scale bar = 50μm. (B) Quantitative analysis of BrdU/Ki67-stained proliferative precursor cells in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P23 (n = 5). The graph shows the fraction of BrdU-labelled cells that lack any positive Ki67 expression 2h after a single BrdU application. Data are mean ± s.e.m.; significant differences are shown in p-value p = 0.0167. (C) Quantitative analysis of BrdU/Ki67-stained proliferative precursor cells in the hilus of CRAF ct (dark bar) and CRAF ko (white bar) at P23 (n = 5). The graph shows the fraction of BrdU-labelled cells that lack any positive Ki67 expression 2h after a single BrdU application. Data are mean ± s.e.m.; significant differences are shown in p-value p = 0.0011. (D) Quantitative analysis of BrdU/Ki67-stained proliferative precursor cells in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P30 (n = 6). The graph shows the fraction of BrdU-labelled cells that lack any positive Ki67 expression 24h after a single BrdU application. Data are mean ± s.e.m.; significant differences are shown in p-value p<0.0001. (E) Quantitative analysis of BrdU/Ki67-stained proliferative precursor cells in the hilus of CRAF ct (dark bar) and CRAF ko (white bar) at P30 (n = 6). The graph shows the fraction of BrdU-labelled cells that lack any positive Ki67 expression 24h after a single BrdU application. Data are mean ± s.e.m.; significant differences are shown in p-value p = 0.0091.</p

CRAF-deficiency in postnatal mice.

<p>(A) Lethality-analysis of CRAF ko embryos around birth (embryonic day 16.5, E16.5-newborn mice at postnatal day 0/1, P0/P1). White bars show total numbers of CRAF ko animals at different developmental stages (E16.5-P0/P1). Dark bars indicate the number of dead mice. No lethal CRAF ko embryos could be observed at E16.5. CRAF ko (E16.5 total), n = 6 (from n = 39 littermates); CRAF ko (E18 total), n = 25 (from n = 145 littermates); CRAF ko (E18 lethal), n = 11, indicating 47% lethality of CRAF ko at E18; CRAF ko (P0/P1 total), n = 109 (from n = 873 littermates); CRAF ko (P0/P1 lethal), n = 73 indicating 67% lethality of CRAF ko at P0/P1. (B) Kaplan-Meier survival curve of postnatal CRAF ct (dark line) and CRAF ko (pointed line) mice. Mice were daily monitored. CRAF ct, n = 763; CRAF ko, n = 110. (C) Western blot analysis of CRAF (C-20) expression in dissected brain areas (hp, hippocampus; pc, prefrontal cortex; cb, cerebellum; bo, olfactory bulb) of postnatal (P30) CRAF^+/+ control (CRAF ct) and CRAF-deficient (CRAF^-/-, CRAF ko) mice. ß-actin serves as loading control. (D) Phenotypic analysis of CRAF ko mice compared to CRAF ct siblings. (Upper line) CRAF ko embryos (right panel) are anyhow smaller and appear more shiny compared to CRAF ct siblings (left panel) at E16.5. (2^nd line) From E18 (left panel) on CRAF ko animals (white arrow) are clearly discernible due to their reduced body size compared to CRAF ct siblings. The placenta is shown on top of animals. (2^nd line, right panel) CRAF ko mice were later born with open eyes (white arrow and Inset). (3^rd line left) Postnatal CRAF ko mice are strongly reduced in body size at the age of P10 (white arrow) with less hair and pink skin color shining through the coat. (3^rd line right) Postnatal CRAF ko mice at the age of P30 showed reduced body size (white arrow) compared to CRAF ct siblings (right). (Lower panel left) Postnatal CRAF ko mice with eye open at birth (EOB) phenotype at the age of P12 compared to CRAF ct sibling with closed eyes. (Lower panel right) CRAF ko mouse at the age of P30 with eyelid defect (white arrow). (E) Quantitative body weight analysis of CRAF ct (dark bars) and CRAF ko (white bars) siblings (n = 234) from postnatal day P1 until P43. Data are mean ± s.e.m.; n = 234. Significant differences are shown in p-value p< 0.05 (*), p< 0.01 (**), p< 0.001 (***). (F) Quantitative brain weight analysis of CRAF ct (dark bars) and CRAF ko (white bars) siblings (n = 234) from postnatal day P1 until P43. Data are mean ± s.e.m.; significant differences are shown in p-value p< 0.05 (*), p< 0.01 (**), p< 0.001 (***). (G) Cerebellar abnormalities in postnatal CRAF ko mice. (Upper panel) Representative sagittal sections of Nissl stained cerebellum of CRAF ct (left) and CRAF ko (right) mice at the age of P30. White arrows in CRAF ko (upper panel right) indicate alterations in lobule X (LX) related to reduced length, failure of proper build central core stream and sulcus compared to CRAF ct (left, white arrows). Also LIII appeared strongly reduced in size in CRAF ko (upper panel right) compared to CRAF ct (left). Scale bars = 1mm. (Lower panel) Representative sagittal sections of Calbindin stained cerebellum LX of CRAF ct (left) and CRAF ko (right) mice at the age of P30. Calbindin-stained Purkinje cell neurons of CRAF ko mice are irregular located in LX. Scale bars = 60μm. Higher magnification of Calbindin-staining reveals elongated and less arborescent dendrite formation of Purkinje cell neurons through the molecular layer. Scale bars = 60μm. (H) Analysis of CRAF expression by immunohistochemistry. Representative sagittal sections of P10 (upper panels) and P30 (lower panels) CRAF ct (left panels) and CRAF ko (right panels) hippocampus stained for CRAF (C-20). White arrowheads indicate CRAF stained cell body of granule neurons in the granule cell layer (GCL) of the hippocampal dentate gyrus in CRAF ct (left panels). CRAF ko sections exhibit no CRAF-expression (right panel). Sections were counterstained with hematoxylin. Scale bar = 50μm.</p

Impaired neuronal differentiation (maturation) of BrdU-labelled NPCs in the DG GCL of postnatal CRAF ko mice.

<p>(A) Immuno-histological analysis of BrdU (green) and the neuronal marker NeuN (red) stained sagittal brain sections of CRAF ct and CRAF ko hippocampus at P35 12 days after a single BrdU application. Representative brain sections of CRAF ct (upper panel) and CRAF ko (lower panel) show BrdU-labelled cells (green) colocalizing with NeuN (red) (merge, white arrows). Scale bar = 50μm. (B) Quantitative analysis of BrdU/NeuN-stained neural precursor cells with neuronal cell fate determination in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P29 (n = 6). The graph shows BrdU-labelled cells that colocalize with NeuN 6 days after a single BrdU application (P23→P29). Analysed cells were normalized with measured GCL Nissl-volume. Data are mean ± s.e.m.; significant differences are shown in p-value p = 0.02. (C) BrdU/NeuN positive cells as a fraction of BrdU-labelled cells in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P29 (n = 6) 6 days after a single BrdU application (P23→P29). Data are mean ± s.e.m.; significant differences are shown in p-value p<0.0001. (D) Quantitative analysis of BrdU/NeuN-stained neural precursor cells with neuronal cell fate determination in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P35 (n = 6). The graph shows BrdU-labelled cells that colocalize with NeuN 12 days after a single BrdU application (P23→P35). Data are mean ± s.e.m.; significant differences are shown in p-value p<0.0001. (E) BrdU/NeuN positive cells as a fraction of BrdU-labelled cells in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P35 (n = 6) 12 days after a single BrdU application (P23→P29). Data are mean ± s.e.m.; significant differences are shown in p-value p<0.0001.</p

Increased numbers of BrdU-labelled NPCs with radial GFAP-positive processes in the DG GCL of CRAF ko at P10.

<p>(A) Immuno-histological analysis of BrdU (green) and the astrocytic marker GFAP (red) stained sagittal brain sections of CRAF ct and CRAF ko hippocampus at P10 24h after a single BrdU application. Representative brain sections of CRAF ct (upper panel) and CRAF ko (lower panel) show BrdU-labelled cells (green) colocalizing with GFAP-positive radial processes (red) (merge, white arrowheads). Scale bar = 50μm. (B) Quantitative analysis of BrdU/GFAP-stained neural precursor cells in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P10 (n = 4). The graph shows BrdU-labelled cells that colocalize with GFAP in radial processes 24h after a single BrdU application. Data are mean ± s.e.m.; significant differences are shown in p-value p = 0.016. (C) BrdU/GFAP positive cells as a fraction of BrdU-labelled cells in the dentate gyrus (DG) GCL of CRAF ct (dark bar) and CRAF ko (white bar) at P10 (n = 4). Data are mean ± s.e.m.; significant differences are shown in p-value p = 0.0224.</p