Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A multi-level approach to understanding the regulation of translation initiation

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis. "September 2016."Includes bibliographical references.mRNA translation is an extremely complex process required for life. Translation consumes vast amounts of cellular resources, and organisms have evolved tight regulatory mechanisms to control this process, which are often deregulated in cancer and other disease states. Initiation, as the rate-limiting step in translation, is particularly well regulated. Two kinase pathways that respond to cellular stresses, the GCN2 and mTORC1 pathways, sense amino acid insufficiency to inhibit translation initiation at distinct points. GCN2 is activated in response to amino acid deprivation and inhibits formation of the ternary complex, comprising elF2, GTP, and the initiator methionyl-tRNA, which is required for recognition of the start codon. Although translation of most mRNAs is greatly suppressed when GCN2 is activated, mRNAs with certain cis elements escape inhibition. In contrast, the mTORC1 pathway is inhibited by the lack of amino acids, which ultimately results in the disruption of eIF4F, a multiprotein initiation factor complex that coordinates the recruitment of the small ribosomal subunit to the 5' end of mRNA. Like a decrease in the amount of ternary complex, disruption of eIF4F also suppresses translation of most mRNAs; however, the translation of a subset of mRNAs harboring a 5'TOP motif is even more dramatically reduced when mTORC1 is inhibited. Here we describe the translational program downstream of amino acid insufficiency, and present evidence of a novel uORF in murine ATF4 whose ribosome occupancy is regulated by the presence of amino acids. We identify the 4EBPs as the mTORC1 substrates that mediate the major effects of mTORC1 inhibition on translation of mRNAs both globally and on 5'TOP mRNAs specifically. Although we cannot mechanistically explain the dependence of 5'TOP mRNA translation on mTORC1 activity, we uncover a surprising role of the cap-proximal sequence in eIF4E recruitment. We systematically assess how the juxtacap sequence modulates eIF4E binding and translation, and present a model whereby the juxtacap sequence dictates the cap-proximal RNA secondary structure in an mRNA-context-dependent manner.by Heather R. Keys.Ph. D

Histidine catabolism is a major determinant of methotrexate sensitivity

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR–Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme—formimidoyltransferase cyclodeaminase—that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.National Cancer Institute (U.S.) (Grant R01 CA129105)United States. Department of Defense (Grant W81XWH-15-1-0337)EMBO Long-Term Fellowship (ALTF 350-2012)American Association for Cancer Research (Grant 16-40-38-KANA)American Cancer Society (Grant PF-12-099-01-TBG)EMBO Long-Term Fellowship (ALTF 1-2014

Genome-wide CRISPR screen for Zika virus resistance in human neural cells

Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention. Keywords: Zika virus; neural progenitors; CRISPR screen; fetal CNS infection; human pluri; potent stem cellsNational Institutes of Health (U.S.) (Grant R01 MH104610)National Institutes of Health (U.S.) (Grant R01 NS088538)National Institutes of Health (U.S.) (Grant U19 AI131135)National Institutes of Health (U.S.) (Grant R33 AI100190)Simons Foundation (Grant SFARI 204106

SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism

One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.National Institutes of Health (U.S.) (Grant R01 CA103866)National Institutes of Health (U.S.) (Grant R01 CA129105)National Institutes of Health (U.S.) (Grant R37 AI47389)United States. Department of Defense (Grant W81XWH-07–0448

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion

A unifying model for mTORC1-mediated regulation of mRNA translation

he mTOR complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses. mTORC1 regulates messenger RNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in mouse cells acutely treated with the mTOR inhibitor Torin 1, which, unlike rapamycin, fully inhibits mTORC1. Our data reveal a surprisingly simple model of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5′ terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1, with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5′ untranslated region length or complexity. mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown. Remarkably, loss of just the 4E-BP family of translational repressors, arguably the best characterized mTORC1 substrates, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin 1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors.National Institutes of Health (U.S.) (Grant CA103866)National Institutes of Health (U.S.) (Grant CA129105)United States. Dept. of Defense (Grant W81XWH-07-0448)W. M. Keck FoundationLAM FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra