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A TPP1 mutation known to cause telomere shortening and bone marrow failure in humans recapitulates telomere loss but results in severe germline defects in mice without impacting murine hematopoiesis., Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.

The establishment of heterochromatin in fission yeast involves methyltransferase Clr4-mediated H3-Lys9 methylation, which is bound specifically by Swi6/HP1. However, the mechanism of propagation of heterochromatin through multiple cell divisions is not known. A role of DNA replication in propagating the heterochromatin is envisaged. Studies inS. pombehave indicated a direct interaction between DNA Polα and Swi6/HP1 and between DNA Polε and Rik1-Dos2 complex, suggesting a coupling between DNA replication and heterochromatin assembly. Here, we show that like DNA Polα, Polδ, which plays a role in both leading and lagging strand replication, also plays a role in silencing at mating type and centromere. We show that both the polymerases α and δ interact directly with both Clr4 and Swi6/HP1. Mutations in both the polymerases lead to decrease in H3-Lys9 methylation and Swi6 at the mating type and left outer repeats of centromeres I and II, with a reciprocal increase in their level at the central element,cnt, at all the three centromeres. These mutations also cause defects in chromosome segregation, recruitment of Cohesin and chromosome dynamics during mitosis and meiosis. Thus, our results indicate that a tight coordination between DNA replication machinery and propagation of the heterochromatin-specific epigenetic mark.

SummaryTelomerase replicates chromosome ends in germ and somatic stem cells to facilitate continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 differing only in 86 amino acids at their N-terminus. While both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates telomere synthesis. We identify a novel intragenic noncoding RNA in the 3’-UTR of the TPP1-encoding gene that specifically shuts down telomerase activation-incompatible TPP1-L to establish TPP1-S as the predominant isoform in somatic cells. Strikingly, TPP1-L is the major isoform in testes, where it can function to restrain telomerase in mature germ cells. Our studies uncover how differential expression of two isoforms allows TPP1 to perform separate functions in different cells, and demonstrate how isoform choice can be determined by an intragenic noncoding RNA.

International audience; The developmental asymmetry of fission yeast daughter cells derives from inheriting older Watson' versus older Crick' DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific imprint', installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase (Pol) has been implicated in the imprinting process. Based on its known biochemical function, Pol might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Pol in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Pol holoenzyme, we find that mutations in the Pol-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Pol and Swi1.

Mammalian chromosomes terminate in stretches of repetitive telomeric DNA that act as buffers to avoid loss of essential genetic information during end-replication. A multiprotein complex known as shelterin prevents recognition of telomeric sequences as sites of DNA damage. Telomere erosion contributes to human diseases ranging from BM failure to premature aging syndromes and cancer. The role of shelterin telomere protection is less understood. Mutations in genes encoding the shelterin proteins TRF1-interacting nuclear factor 2 (TIN2) and adrenocortical dysplasia homolog (ACD) were identified in dyskeratosis congenita, a syndrome characterized by somatic stem cell dysfunction in multiple organs leading to BM failure and other pleiotropic manifestations. Here, we introduce the biochemical features and in vivo effects of individual shelterin proteins, discuss shelterin functions in hematopoiesis, and review emerging knowledge implicating the shelterin complex in hematological disorders.

Summary: Telomerase replicates chromosome ends in germ and somatic stem cells to facilitate their continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here, we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 that appear to be generated from separate transcripts and differ only in 86 amino acids at their N terminus. Although both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates efficient telomere synthesis. We find that TPP1-S is the predominant isoform in somatic cells, and strikingly, TPP1-L is the major isoform in differentiated male germ cells. We observed that TERT expression persists in these germ cells, suggesting that TPP1-L could restrain telomerase in this context. We show how differential expression of TPP1 isoforms determines telomerase function and demonstrate how alternative transcription start sites allow one gene to perform distinct functions in different biological contexts. : Human TPP1 is critical for telomerase function. Grill et al. demonstrate that TPP1 exists as two isoforms: TPP1-S and TPP1-L. TPP1-S, but not TPP1-L, activates telomerase. TPP1-S is the major isoform in all somatic cells, and TPP1-L is upregulated in differentiated germ cells to presumably curb telomerase that persists there. Keywords: telomerase, TPP1, somatic cell, stem cell, germ cell, cancer

The developmental asymmetry of fission yeast daughter cells derives from inheriting “older Watson” versus “older Crick” DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific “imprint”, installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The biochemical nature of the imprint and the mechanism of its installation are still not understood. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install themat1imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a role of Polα in nicking through putative endonuclease domains but confirm its role in installing an alkali-labile moiety as the imprint. A detailed genetic and molecular analysis reveals a direct role of the Cdc23/Mcm10 primase activity in installing the imprint in cooperation with Polα and Swi1.

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci.

Background: Organotypic culture models developed using 3D conditions recapitulate tissue-specific structural features and cell-cell interactions more accurately than conventional 2D cultures. Our ultimate goal is to optimize culture conditions which promote the survival and proliferation of multiple myeloma (MM) cells and could serve as a platform for molecular mechanistic, clinical biomarker and pharmacodynamic marker studies using immune-modulatory compounds (IMIDs) and other myeloma drugs alone and in combination. Design/Results: Using gas permeable microfluidic devices, we cultured and compared growth/morphologic properties of six multiple myeloma cell lines, MM1.S, MM1.SPR, H929, H929PR, H929-220R and RPMI-8226 in 2D and 3D conditions. Collagen type IV was used as an extra-cellular matrix source to grow these cells. Cell growth and morphology was captured at regular intervals. Ten days post culture, cells were harvested from the device and stained for proliferation (Ki67 staining) index and expression of key MM oncogenic molecules, CD138, CD38 and BCMA. Cell lines grown in 3D conditions had, with some exceptions, higher proliferation index compared to 2D conditions. Thus, Ki67-mean fluorescence intensity (MFI) for 3D vs 2D were: 2038 vs 1130 for MM1.S; 1614 vs 1912 for MM1.PR; 2067 vs 1169 for H929; 2057 vs 1702 for H929PR; 2300 vs 1889 for H929-220R; 2018 vs 1220 for RPMI-8226. Similar trends for higher proliferation under 3D conditions were observed for the CD138, CD38 and BCMA cell subsets. Expression of FOXM1, a potential marker of IMID resistance, was reduced in Pomalidomide sensitive non-synchronous cells compared to resistant cells, although a few clusters with higher FOXM1 expression were observed among sensitive cells. To further study the effects of other components of MM tumor micro-environment on Pomalidomide response, we optimized the culture conditions to co-culture MM cell lines with bone marrow stromal cells. The co-culture of bone marrow stromal cells, HS5 with MM cell line H929 protected Ikaros degradation induced by Pomalidomide. Interestingly, CD44 expression in H929 cells was upregulated in co-culture conditions with stromal cells. Future Directions: These culture conditions are currently being optimized to study the (1) drug effects in MM and immune cells alone and in combination and (2) use the co-culture derived cells for single cell level evaluation of genetic, transcriptomic or proteomic changes associated with drug treatment and (3) ultimately grow primary Myeloma cells in these conditions for ex vivo manipulation and downstream molecular and biological effects. Figure. Figure. Disclosures Ahsan: celgene: Employment, Equity Ownership. Jeyaraju:Celgene Corporation: Employment, Equity Ownership. Bisht:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Pierceall:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed.

Sister chromatid cohesion relies on cohesin, a complex comprised of a tri-partite ring and a peripheral subunit Scc3, which is found as two related isoforms SA1 and SA2 in vertebrates. There is a division of labor between the vertebrate cohesin complexes; SA1-cohesin is required at telomeres and SA2-cohesin at centromeres. Depletion of SA1 has dramatic consequences for telomere function and genome integrity, but the mechanism by which SA1-cohesin mediates cohesion at telomeres is not well understood. Here we dissect the individual contribution of SA1 and the ring subunits to telomere cohesion and show that telomeres rely heavily on SA1 and to a lesser extent on the ring for cohesion. Using chromatin immunoprecipitation we show that SA1 is highly enriched at telomeres, is decreased at mitosis when cohesion is resolved, and is increased when cohesion persists. Overexpression of SA1 alone was sufficient to induce cohesion at telomeres, independent of the cohesin ring and dependent on its unique (not found in SA2) amino terminal domain, which we show binds to telomeric DNA via an AT-hook motif. We suggest that a specialized cohesion mechanism may be required to accommodate the high level of DNA replication-associated repair at telomeres.

Heterochromatin assembly in fission yeast is initiated by binding of Swi6/HP1 to the Lys-9-dimethylated H3 followed by spreading via cooperative recruitment of Swi6/HP1. Recruitment of Cohesin by Swi6/HP1 further stabilizes the heterochromatin structure and integrity. Subsequently, polyubiquitylation of Cut2 by anaphase-promoting complex-cyclosome (APC/C)-ubiquitin-protein isopeptide ligase (E3 ligase) followed by degradation of Cut2 releases Cut1, which cleaves the Rad21 subunit of Cohesin, facilitating sister chromatid separation during mitosis. Here, we demonstrate a surprising role of APC/C in assembly of heterochromatin and silencing at mating type, centromere, and ribosomal DNA loci. Coincidentally with the loss of silencing, recruitment of Swi6, H3-Lys-9-Me2, and Clr4 at dg-dh repeats at cen1 and the K region of mat locus is abrogated in mutants cut4, cut9, and nuc2. Surprisingly, both Cut4 and Cut9 are also highly enriched at these regions in wild type and depleted in swi6Δ mutant. Cut4 and Cut9 interact directly with Swi6/HP1 and Clr4, whereas the mutant Cut4 does not, suggesting that a direct physical interaction of APC subunits Cut4 and Cut9 with Swi6 and Clr4 is instrumental in heterochromatin assembly. The silencing defect in APC mutants is causally related to ubiquitylation activity of APC-E3 ligase. Like swi6 mutant, APC mutants are also defective in Cohesin recruitment and exhibit defects like lagging chromosomes, chromosome loss, and aberrant recombination in the mat region. In addition, APC mutants exhibit a bidirectional expression of dh repeats, suggesting a role in the RNA interference pathway. Thus, APC and heterochromatin proteins Swi6 and Clr4 play a mutually cooperative role in heterochromatin assembly, thereby ensuring chromosomal integrity, inheritance, and segregation during mitosis and meiosis.