HP6/Umbrea is dispensable for viability and fertility, suggesting essentiality of newly evolved genes is rare.

The newly evolved gene Heterochromatin Protein 6 (HP6), which has been previously classified as essential, challenged the dogma that functions required for viability are only seen in genes with a long evolutionary history. Based on previous RNA-sequencing analysis in Drosophila germ cells, we asked whether HP6 might play a role in germline development. Surprisingly, we found that CRISPR-generated HP6 mutants are viable and fertile. Using previously generated mutants, we identified an independent lethal allele and an RNAi off-target effect that prevented accurate interpretation of HP6 essentiality. By reviewing existing data, we found that the vast majority of young genes that were previously classified as essential were indeed viable when tested with orthologous methods. Together, our data call into question the frequency with which newly evolved genes gain essential functions and suggest that using multiple independent genetic methods is essential when probing the functions of young genes.

Molecular mechanisms of telomere biology disorders.

Genetic mutations that affect telomerase function or telomere maintenance result in a variety of diseases collectively called telomeropathies. This wide spectrum of disorders, which include dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, is characterized by severely short telomeres, often resulting in hematopoietic stem cell failure in the most severe cases. Recent work has focused on understanding the molecular basis of these diseases. Mutations in the catalytic TERT and TR subunits of telomerase compromise activity, while others, such as those found in the telomeric protein TPP1, reduce the recruitment of telomerase to the telomere. Mutant telomerase-associated proteins TCAB1 and dyskerin and the telomerase RNA maturation component poly(A)-specific ribonuclease affect the maturation and stability of telomerase. In contrast, disease-associated mutations in either CTC1 or RTEL1 are more broadly associated with telomere replication defects. Yet even with the recent surge in studies decoding the mechanisms underlying these diseases, a significant proportion of dyskeratosis congenita mutations remain uncharacterized or poorly understood. Here we review the current understanding of the molecular basis of telomeropathies and highlight experimental data that illustrate how genetic mutations drive telomere shortening and dysfunction in these patients. This review connects insights from both clinical and molecular studies to create a comprehensive view of the underlying mechanisms that drive these diseases. Through this, we emphasize recent advances in therapeutics and pinpoint disease-associated variants that remain poorly defined in their mechanism of action. Finally, we suggest future avenues of research that will deepen our understanding of telomere biology and telomere-related disease.

A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

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.

TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis.

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1's functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.

Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells.

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.

The N Terminus of the OB Domain of Telomere Protein TPP1 Is Critical for Telomerase Action.

Telomerase recruitment to telomeres and enzymatic processivity are mediated by TPP1, an essential component of telomere integrity and telomerase function. A surface on the OB domain of TPP1 called the TEL patch is critical for TPP1's telomerase-associated functions. Here, we identify a separate region in the N terminus of the OB domain (termed NOB) of TPP1 that, like the TEL patch, is essential for telomerase repeat addition processivity in vitro as well as telomerase recruitment to telomeres and telomere lengthening in cells. Although well-conserved among most mammalian TPP1 homologs, the NOB region in mice is distinct. Swapping the sequence of human NOB into mouse TPP1 allows it to stimulate human telomerase, qualifying NOB as an important determinant of species specificity for TPP1-telomerase interaction. Our studies show that TPP1 NOB is critical for telomerase function and demonstrate that the telomerase interaction surface on TPP1 is more elaborate than previously appreciated.