What I have read of interesting papers this week

Some useful reviews

One in Nature Medicine “Refining strategies to translate genome editing to the clinic” (Cornu et al. 2017). It covers some of the recent gene editing clinical trials and outlines key standardisations required to translate CRISPR and similar techniques into effective therapeutic strategies.

Brendand Manning and Alex Toker have written a comprehensive Cell review “AKT/PKB Signaling: Navigating the Network”. Looking forward to giving it a proper read!

Insulin signalling

Key paper in Cell: The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover (Tawo et al. 2017)

Apparently, previous studies have shown that although CHIP-null mice exhibit normal embryonic development, lack of CHIP (ubiquitin ligase, key for cellular protein quality control) accelerates ageing. Also, the livers of these mice develop insulin resistance. Using C. elegans and D. melanogaster, this study demonstrates that under non-stress conditions, CHIP ubiquitylates INSR and targets it for endocytic (not proteasomal) degradation; however, under proteotoxic stress, CHIP is “busy” sorting out other damaged proteins and this results in increased INSR levels and enhanced downstream activation of PI3K/AKT signalling. Interestingly, they make a link to severe INSR due to mutations in the receptor by mentioning K1095E; apparently, they have identified this residue to be ubiquitylated. So the findings are extrapolated to speculations about the effects of metabolic insults on INSR degradation and therefore glucose homeostasis.

Metabolic

Chad Cowan and co. on “Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease” (Friesen et al. 2017, Stem Cell Reports)

From Jens Brüning’s lab on IL-6: IL-6 improves energy and glucose homeostasis in obesity via enhanced central IL-6 trans-signaling (Timper et al. 2017 Cell Reports) – not read in detail, but abstract suggests that similar to leptin, central IL-6 signalling suppresses feeding and improves glucose tolerance, but its actions are actually enhanced upon challenging mice with HFD. And the mechanism of signalling is interesting – the soluble IL-6R complexes with gp130 on the target cell surface, hence why they call this trans-signalling.

Obesity-induced hepatic steatosis is mediated by endoplasmic reticulum stress in the subfornical organ of the brain (Horwath et al. 2017, JCI Insight) – not read beyond abstract, but might be of relevance. Apparently reporting an uncoupling of hepatic steatosis from other obesity-linked conditions – i.e. reducing brain stress can reverse NAFLD, but without altering body weight, food intake, adiposity, or hypertension.

β-Klotho deficiency protects against obesity through a crosstalk between liver, microbiota, and brown adipose tissue (Somm et al. JCI Insight) – also only given this one a brief skim of the abstract and the introduction.

Some “old” stem cell signalling knowledge

An 10-year-old paper, but nonetheless interesting for me…

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling (Wang et al. 2007, Blood)

The abstract of this paper rightly starts out by stating that little is known about the cell-surface receptors (and implicit signalling pathways) that are activated under conditions that support stem cell self-renewal – despite improvements in developing defined conditions. Advances have been made in the past 10 years, but I would still argue that a lot remains to be learned.

The hESCs used in this study are maintained in MEF-conditioned medium on Matrigel or in defined medium supplemented with Heregulin-1B, Activin A, IGF1 & FGF2. Following various stimulations, the cell lysates are profiled with RTK arrays (42 human RTKs) to identify activated receptors. Hits are subsequently validated with inhibitor studies.

There is a strong phosphorylation of IGF1R and IR upon addition of conditioned medium, and the study then goes on to determine that it is IGF1R specifically that maintains self-renewal and prevents differentiation in stem cell maintenance conditions. Interestingly, they find that the stem cells express high levels of IGF1R (flow cytometry) and very low expression of insulin receptor (IR), which is consistent with my observations of absence of a response to insulin stimulation, but a clear activation of PI3K/AKT signalling in response to IGF1 stimulation. Looks quite solid and they also show a requirement for ERBB2/3 signalling. One thing that surprises me, though, is that they perform GF-depletion studies (DMEM/F12 + 0.5 % BSA) overnight which is really harsh for stem cells – mine start to day after 3h.

Another study was published in Nature at the same time as this paper, supporting the notion that IGF1R signalling in stem cells is essential for their self-renewal potential (Bendall et al. 2007, Nature).

Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of StemCell Differentiation (Petruk et al. 2017a, Molecular Cell) – there is an accompanying paper by the same group Petruk et al. 2017b in Cell Reports as well.

This is quite cool, by the group who originally developed a chromatin assembly assay relying on PLA (proximity ligation assay) to study the structure of nascent chromatin. Here, they show that immediately following DNA replication in stem cells, there is a delay in H3K27me3 deposition on the DNA, which allows transcription factors induced by differentiation signals to bind to their target sites. This is a very finely tuned time window that allows for exquisite control of differentiation only when the right transcription factors are induced and when the chromatin is open. Because the time window is limited, it also prevents spurious binding of other transcription factors later on, essentially allowing the cell to reconfigure itself in the process of differentiation once the primary transcription factors have done their job. There is an accompanying news and views as well: A Determined “Hesitation” on H3K27me3 Empowers Stem Cells to Differentiate (Huang and Wang 2017, Molecular Cell). In the Cell Reports paper, which I haven’t read, they apparently show that the same is true for HSCs.

Of general interest

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity (Saleheen et al. 2017, Nature)

Moving beyond non-human models to understand the complete  loss of certain genes in human. This is possible due to large scale sequencing of inbred populations that are likely to harbour two copies of rare mutations that result in complete loss of function for a particular gene. Really interesting and a more comprehensive catalogue of possible human knockouts is soon to come out. In this study, the authors link the discovered gene knockouts to more than 200 phenotypic traits, providing a detailed functional analysis. Interesting to go back and link the phenotypes to those observed in mice engineered to lack some of the same genes – you might be surprised at some of the discrepancies, cautioning us against thinking of mice as mini-humans. An accompanying news and views is also available at Nature.

 

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency (Yang et al. 2017, Cell)

This is quite ground-breaking in the developmental world (if it stands the test of independent replication). The study reports the first ever derivation of pluripotent stem cells (both mouse and human) with both extraembryonic (i.e. can generate placenta etc.) and embryonic capacity, terms extended pluripotent stem (EPS) cells.

Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation (Slobodin et al. 2017, Cell)

Quite cool both on the biological and techy side of things. Very, very nice approach to screen for differential promoter effects by using a barcoded reporter, essentially designing a new technique “Barcoded polysomal profiling” (BPP). Biologically, the study reports that mA6 of RNA is an epigenetic mechanism that determines the future activity and fate of the transcribed mRNA, thereby indirectly coupling transcription and translation.

Not read in detail, but this sounds a bit cool (apparently, you can change the sex of C. elegans with fatty acids): Fatty Acids Regulate Germline Sex Determination through ACS-4-Dependent Myristoylation (Tang et al. Cell 2017 quite impressive with only 2 authors!).

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a (Swarts et al. 2017, Molecular Cell –  from the Jinek lab) – just one of those papers that are good to have; Cas12a = Cpf1, the cousin of Cas9, also used for gene editing. Different from Cas9 by being able to process its own guide RNAs + different PAM requirement + Cpf1 generates staggered cuts.

 

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Science this week

Stem cell research

Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells (Avior et al. 2017, Stem Cell Research)

I love reading good papers reporting modelling of early-onset human diseases in human stem cells. There is one in Stem Cell Research that caught my attention this week, by Avior et al., modelling ablation of retinoblastoma protein (Rb) in human embryonic stem cells (hESCs). This is a well-known tumour suppressor and an inherited loss of Rb protein leads to retinoblastoma development, the most common primary intraocular pediatric cancer (95 % of cases diagnosed before the age of 5). In 6 % of cases, patients develop trilateral retinoblastoma characterised by neuroectodermal tumour occurence in additional to retinal tumours in the eye. Combined with results from conditional knockout mice, the Rb protein has been suggested to play a role in neural development. In the current study,  following CRISPR/Cas9 gene editing to knockout the Rb protein, modified hESCs are subjected to an array of assays to study the resulting phenotype. After observing normal neural stem cell generation in a 2D directed differentiation protocol, despite expectations of a phenotype compared to wildtype hESCs, the authors conclude that a more complex differentiation paradigm is needed. As a result, they move on to generate teratomas in immunocomprised mice and observe a substantial expansion of neural structures in Rb-null teratomas. This highlights the importance of using multiple methods to assess a phenotype reliable. I have come to the same conclusion in my studies as 2D differentiation protocols often rely on non-physiological concentrations of various drugs and peptides, whereby a phenotype may remain masked. This study also reveals mitochondrial dysfunction in Rb-null hESCs and establishes a framework for high-throughput testing of hundreds of FDA-approved chemotherapies in this human cell models.

 

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells (D’Antonio et al. 2017, Stem Cell Research)

This paper made me aware of fluorescent cell barcoding as an efficient way to pool multiple stem cell samples together and save on antibodies during initial characterisation. To make pluripotency and multilineage analysis across multiple stem cell lines available, the authors provide an Excel file which users can use to generate their own scores and heatmaps using Ct values of a reference gene of interest and any chosen marker genes.

Another paper from the same lab: Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics (Panopoulus et al. 2017 Cell Stem Cell)

A lot of effort is invested into understanding how iPSCs differ from hESCs and whether somatic memory may limit their utility in disease modelling and as potential treatment strategies. It is not well understood whether aberrant methylation of CpG sites in iPSCs is mainly caused by genetic variability or are artefacts of the reprogramming factors. The current study reports the following: “Here we generated 22 iPSC clonal lines from six individuals (three pairs of older monozygotic twins). We profiled the 22 iPSC lines, at early (passages 5 [p5] and 9 [p9]) and late (passage 20 [p20]) passages, as well as fibroblasts (tissue of origin) using genome-wide methylation arrays and RNA sequencing (RNA- seq) data.We estimate aberrantmethylation of the iPSCs relative to ESCs, and we show that aberrant methylation affects gene expression and is enriched for CpGs associated with MYC and MYC-related protein motifs. We then identify genome-wide associations between CpG methylation variation and genetic background, clone, and passage, andwe showthat these associ- ationslikely result fromrelevant biological processes.Weexamine whether aberrant CpGs are enriched for CpGs associated with genetic and non-genetic effects, and we show that aberrant methylation preferentially occurs at CpGs showing clone-associ- ated effects and is less enriched at sites associated with genetic background. Our study shows that non-genetic regulatory mechanisms associated with clone-specific effects most strongly underlie iPSC aberrancy.”

My immediate reservations without having read the study in detail is the limited number of twin pairs and therefore cells examined as well as the fact that they are all female.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types (Panopoulus et al. 2017 Stem Cell Research)

An iPSC resource from the same lab  (very productive!). Will be useful for examining phenotypic diversity across individual induced pluripotent stem cell lines.  Pertinent to the same topic, a study in Cell Stem Cell examines the influence of genetic variation on gene expression in human iPSCs: Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells (DeBoever et al. 2017 Cell Stem Cell). Actually, this seems to be a theme in the current Cell Stem Cell issues and multiple other papers examine the same topic from different perspectives. For instance, Warren et al. provide proof-of-concept that large cohorts of human iPSCs can be used to performed GWAS studies in a dish, here in the context of metabolic disease.

 

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells (Guo et al. 2017 Protein & Cell)

A variation on a system that has been introduced in the past – the ability to activate genes from their endogenous locus via a Dox-inducible expression of dCas9 fused to a transcriptional activation domain.

Other bits and pieces that I have not read in detail, but that caught my interest – mostly metabolism-related

Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling (Bertholet et al. 2017 Cell Metabolism) – up until now all beige adipocytes were assumes to function like brown adipocytes, utilising UCP1 dissipate energy as heat; this paper suggests that there is a subset of beige adipocytes in mice, in epididymal fat in particular, that are UCP-negative yet perform thermogenesis by engaging in futile creatine cycling. Quite biophysical, by the way! If of interest, there is a Preview commentary on this article as well: Now UCP(rotein), Now You Don’t: UCP1 Is Not Mandatory for Thermogenesis (Szabo and Zoratti 2017, Cell Metabolism).

Another paradigm-shaking paper in Cell Metabolism modifies our understanding of the incretin effect mediated by GLP1. I have actually only read the Preview by Habener and Stanojevic (Pancreas and Not Gut Mediates the GLP-1-Induced Glucoincretin Effect) and it is always best to have a look at the actual paper and its data instead, so acknowledging the absence of appropriate assessment on my own, it might still be worth a read for those interested in this field!

Surprising results are the theme in Cell Metabolism this week; another one: FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms by BonDurant et al., providing evidence that adiponectin is dispensable for the metabolic effects of FGF21.

A paper in PNAS by Steptoe and Wardle (Life skills, wealth, health, and wellbeing in later life) looks at the importance of 5 key life skills – conscientiousness, emotional stability, determination, control, and optimism – in later life (cross-sectionally and longitudinally), concluding that (after adjustment of multiple confounding factors) these life skills are associated with wealth, income, subjective wellbeing, less depression, low social isolation and loneliness, more close relationships, better self-rated health, fewer chronic diseases and impaired activities of daily living, faster walking speed, and favorable objective biomarkers (concentration of high-density lipoprotein cholesterol, vitamin D and C-reactive protein, and less central obesity). They were also associated with greater psychological well-being and less loneliness, and a lower incidence of new chronic disease and physical impairment over a 4-y period. I like this study – a good reason to practice mindfulness throughout life!

 

 

 

 

The weekly science round up – 5 April

Actually, some of  the papers already came out last week, but only just got round to having a proper look at them.

Science

Lysosomal cholesterol activates mTORC1 via an SLC38A9–Niemann-Pick C1 signaling complex (Castellano et al.)

Copied from the final bit of the paper: “This work identifies dedicated machinery that couples cholesterol trafficking through the lyso- some to regulation of cellular growth signaling. LDL-derived cholesterol affects mTORC1 through the combined action of a positive regulator, SLC38A9, and a negative regulator, NPC1 (Fig. 4G). SLC38A9 conveys increases in lysosomal cholesterol through its conserved CARC and CRAC motifs, leading to lysosomal recruitment and activation of mTORC1. In contrast, NPC1 associates with the mTORC1 scaffolding complex and translates cholesterol depletion into mTORC1 inhibition.”

Nature

Structural insights into adiponectin receptors suggest ceramidase activity (Vasiliauskaité-Brooks et al.)

Discovery that ADIPORs (1+2) have ceramidase activity that can be enhanced upon adiponectin stimulation. Overall, this enzymatic activity is low so future studies are required to understand whether it bears relevance to the function of adiponectin in vivo and its link to metabolic improvements.

 

The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue (Lynes et al.) 

Cold-induced activation of brown adipose tissue (BAT) is known to result in increased energy expenditure, and it is believed that BAT energy expenditure can be harnessed therapeutically in metabolic disorders. Using global lipodmics, this study identifies a BAT-specific lipokine 12,13-diHOME which is released in response to cold exposure and activates BAT fuel utilisation. Consistent with a decrease in BAT mass with obesity, 12,13-diHOME levels were negatively correlated with obesity and other phenotypes characteristic of the metabolic syndrome. It is impressive that this lipokine is first identified in human participants exposed to cold, followed by mechanistic studies in mouse models, where the effects of 12,13-diHOME on free fatty acid uptake are comparable to noradrenaline-mediated BAT activation. Overall, an elegant study advancing the field with a new candidate for pharmacologic BAT activation.

 

Nature Biotechnology

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells (Weinberg et al.)

My brain gets mushy when I try to read this in detail at 10 pm in the evening, but just quite cool to see the engineering of one of the most sophisticated computational biological circuits to date. This done by combining orthogonal recombinases and various DNA sequences that control transcription. Again, I think an electrical engineer would understand this paper better than me, but still impressive…

Synergistic drug combinations for cancer identified in a crisPr screen for pairwise genetic interactions (Han et al.)

In this paper, a global CRISPR screen is performed to identify combinatorial interactions between cancer drug targets. 490,000 sgRNAs are used to screen for synthetic lethal drug target pairs in K562 leukemia cells. The approach is interesting and applicable to other areas. Worth noting the technical aspects of the screen.

A similar paper has also been published in Nature Methods: Combinatorial CRISPR–Cas9 screens for de novomapping of genetic interactions (Shen et al.)

Nature Protocols

From the Zhang lab on CRISPR screens: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening (Joung et al.)

 

Nature Genetics

Estimating the selective effects of heterozygous protein-truncating variants from human exome data (Cassa et al.)

Not the best person to understand this, but a study that looks at protein truncating variatns in ExAC if they are predicted to be consequential and estimates gene-based fitness effects and individual gene fitness cost in heterozygotes (only using data from individuals with non-Mendelian disorders). Basically useful to look at novel genes that might have important functions in development as mutations would be highly selected against. The mTOR and MAPK pathways are in the list of pathways with genes that seem to have a high gene fitness cost (unsurprising).

Nature Chemical Biology

Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase (Stanford et al.)

Worth a read – actually quite metabolic and signalling-relevant. Here is the abstract:

“Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.”

JCI Insight

Insulin’s direct hepatic effect explains the inhibition of glucose production caused by insulin secretion (Edgerton et al.)

Given the redundancy in insulin’s ability to control hepatic glucose production (direct and indirect mechanisms), this study set out to explore the contextual importance of some of these mechanisms.

An interesting excerpt from the introduction of the paper, discussing the physiological difference between peripheral insulin infusion and direct infusion into the portal vein: “It is apparent, therefore, that when insulin is infused into a peripheral vein its direct effects on the liver are underemphasized, while its indirect effects are exaggerated. Thus, the route of insulin delivery can have a major impact on both the overall response of the liver and the mechanisms by which that response is achieved. Indeed, over a range of insulin doses, peripheral insulin infusion was not as effective at suppressing HGP as compared with direct infusion into the portal vein.”

Based on this information, “the purpose of this study was to assess whether insulin’s acute indirect effects on HGP are additive to, redundant to, or synergistic with its direct hepatic effects, in the context of a physiologic increase in portal vein insulin level in a large animal model. The impact of eliminating each of insulin’s indirect effects, either alone or in combination, was determined. We focused on insulin’s ability to lower FFAs, activate brain insulin signaling, and reduce glucagon secretion since recent studies have concluded that suppression of lipolysis is the major mechanism by which insulin suppresses HGP (11), that increased brain insulin action is required for the rapid suppression of HGP (17), and because a fall in plasma gluca- gon is potentially a powerful contributor to insulin’s ability to inhibit HGP (15).”

They perform portal insulin infusion (in mice) and simulate a 6-fold increase in insulin secretion, but manage to maintain the physiologic insulin gradient between liver and the rest of the body.

This paper is very interesting given the multiple demonstrations of a key role of insulin’s indirect effect on HGP via modulation of FFA release from adipose tissue; in contrast, this study seems to imply that FFA don’t play a role in HGP suppression under what is described as more physiologic conditions. Similarly, a fall in glucagon secretion is not necessary for insulin direct ability to suppress HGP. The same is true when insulin’s brain action is inhibited. The discussion is really worth a read!

EMBO Journal

AKT-phosphorylated FOXO1 suppresses ERK activation and chemoresistance by disrupting IQGAP1-MAPK interaction (Pan et al.)

Looking forward to reading this one at some point! The accompanying News and Views sums it all up: “And Akt-ion! IQGAP1 in control of signaling pathways” (Choi et al.

Mfn2 is critical for brown adipose tissue thermogenic function (Boutant et al.)

Worth a read! Adipose tissue-specific KO of Mfn2 results in increased fat accumulation in BAT even on a low-fat diet as well as an overall decrease in energy expenditure and an inability to sustain normal body temperature via cold-induced BAT thermogenesis. This is linked to an impaired O2 flux in BAT when specifically measuring the different complexes forming the electron transport chain. At the protein level, Complex I is almost absent upon Mfn2 ablation. Mfn2 is shown to interact with perilipin and the data suggest that this enhances mitochondria-lipid droplet interactions. It seems that glycolysis is increased due to mitochondrial dysfunction. There is also an increased accumulation of FAT in subcutaneous WAT and the authors suggest that this exlpains the improved metabolic profile of HFD-fed adipose-specific Mfn2 KO mice.

USP49 negatively regulates tumorigenesis and chemoresistance through FKBP51-AKT signaling (Luo et al.)

Had a quick skim – in cancer cell lines and cancer mouse models, but the mechanism might be valid in a more physiological context, too. Basically, USP49 deubiquinates FKBP51 which results in increased stabilisation. In turn, FKBP51 promotes the interaction between AKT and PHLPP, which dephosphorylates AKT at S473 thereby reducing its activation.

The phosphorylation status of T522 modulates tissue-specific functions of SIRT1 in energy metabolism in mice (Lu et al.)

Just skimmed the abstract, but might be interesting to adipogenesis enthusiasts.

eLife

Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis (An et al.) – only read abstract, but might be relevant. Just in press, so still not formatted properly, i.e. annoying to read.

Diabetes

 

 

Other exciting bits

I have been reading a lot of developmental biology recently and came across the following Guardian coverage of the first experiments that make it possible to break the 14-day rule for embryo research. I just find the videos and images amazing + the useful infographic of lineage specification at the bottom. The two papers giving rise to the Guardian feature can be found at Nature and Nature Cell Biology. These are landmark studies and were recently followed up by a study from the same Cambridge group, now demonstrating that embryonic and extra-embryonic stem cells can be assembled in vitroto mimic embryogenesis (effectively generating embryos in vitro, see Harrison et al. 2017 Science).

In terms of interesting metabolism reviews, it is worth having a look at Nature Reviews Endocrinology – there are good ones on adipose tissue etc.